CN102762096A - Modified cry1ca insecticidal cry proteins - Google Patents

Modified cry1ca insecticidal cry proteins Download PDF

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CN102762096A
CN102762096A CN2010800640116A CN201080064011A CN102762096A CN 102762096 A CN102762096 A CN 102762096A CN 2010800640116 A CN2010800640116 A CN 2010800640116A CN 201080064011 A CN201080064011 A CN 201080064011A CN 102762096 A CN102762096 A CN 102762096A
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albumen
seq
dig
amino acid
toxin
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T.米德
K.纳瓦
T.海伊
I.拉里努阿
A.T.伍斯利
S.L.伯顿
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Corteva Agriscience LLC
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Dow AgroSciences LLC
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The present invention includes modified, insecticidal B.t. Cry1Ca proteins, including the proteins designated herein as DIG-109 and DIG-152, as well as variants of DIG-109 and DIG-152, nucleic acids encoding these proteins, methods of controlling pests using the proteins, methods of producing the proteins in transgenic host cells, and transgenic plants that produce the proteins. The DIG-109 and DIG-152 proteins comprise chimeric peptides composed of a core toxin segment of B.t. Cry1Ca and a Cry1Ab protoxin segment. Insecticidally active variants of the DIG-109 and DIG-152 proteins are also described.

Description

The CRY1CA desinsection CRY albumen of modifying
Invention field
The present invention relates to new Cry insecticidal proteins and be used for the purposes of Pest Control.
Background of invention
Autumn mythimna separata [Fall armyworm, FAW; Noctuid (Spodoptera frugiperda) is coveted on the meadow] cause corn and other crops grievous injury of soybean and cotton for example.
(Bacillus thuringiensis B.t.) is a kind of bacterium of soil media to bacillus thuringiensis, and it produces insecticidal crystal protein, and the latter is called as δNei Dusu or Cry albumen.Cry albumen is a mouthful toxicity, and it works through the midgut epithelial cells to the susceptible insect and brings into play function.Also upgrade the tabulation of a large amount of δNei Dusus: lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html regularly in this website maintenance.
Express the transgenic corns, particularly Cry1F of the gene of coding Cry albumen, the anti-FAW effect of commercial level is provided.
Although FAW resistant transgenic corn is succeedd, the possibility of resistant insects population development is still threatening the long-term durability of Cry albumen in FAW control, and brings discovery and develop the demand of new Cry albumen with control FAW and other insect.There is the insect of resistance can develop (Heckel et al., 2007, Pigott and Ellar, 2007) to B.t.Cry albumen through several mechanisms.In insect, identify the multiple receptor protein classification of Cry albumen, and had multiple instance in every kind of acceptor classification.The sudden change of toxin bound fraction of cadherin domain that maybe be through receptor protein to the resistance of popular Cry albumen develops.The further method of resistance is to handle protease through parent toxin to mediate.Thereby the resistance to the Cry toxin in the lepidopterous species has complicated genetics basis, has at least 4 kinds of different, main resistant genes.Lepidopterous insects to Cry albumen resistance grows up in following scope: diamond-back moth (Plutella xylostella) (Tabashnik; Et al.; 1994), cabbage looper (Trichoplusia ni) (Janmaat and Myers 2003,2005); And Heliothis zea (Helicoverpa zeae) (Tabashnik et al., 2008).Developing new high potentiality Cry albumen will provide the additional means of management FAW and other insect.The Cry albumen with dissimilar effects of in transgenic corns, uniting generation will limit the development of FAW insect-resistant, and the long-term effectiveness of protection B.t. technology in insect control.
Summary of the invention
The invention provides desinsection B.t.Cry albumen; Comprise that this paper is appointed as the albumen of DIG-109 and DIG-152; And the variant of DIG-109 and DIG-152, the nucleic acid of these albumen of encoding is with the method for these albumen Pest Control; In genetically modified host cell, produce the method for these albumen, and the genetically modified plants that produce these albumen.
Of embodiment 1, DIG-109 and DIG-152 albumen comprise the chimeric peptide of being made up of B.t.Cry1Ca active toxin fragment and Cry1Ab parent toxin fragment.The insecticidal activity variant of DIG-109 and DIG-152 albumen has also been described.
To be DIG-109 and DIG-152 albumen have activity to the autumn armyworm larvae and the sugarcane moth borer larva population of Cry1F resistance in a surprising discovery of this paper report.Thereby DIG-109 and DIG-152 albumen are the ideal candidates persons who is used to control lepidoptera pest.This albumen can be separately or with other Cry albumen Cry1F for example, Cry1Ab and Cry1Ac unite use, with the development of control resistant insects population.For the discussion of these insects, referring to for example Tabashnik, PNAS (2008), vol.105 no.49,19029-19030.
The insecticidal activity fragment of DIG-109 and DIG-152, and the nucleotide of these fragments of encoding are another aspect of the present invention.
In an embodiment, the invention provides the DIG-109 polypeptide of separation, comprise and be selected from following active toxin fragment:
The polypeptide of amino acid sequence that comprises the 28-619 residue of SEQ ID NO:1;
Comprise the polypeptide that has with the amino acid sequence of the amino acid sequence at least 90% sequence homogeneity of the 28-619 residue of SEQ ID NO:1;
Comprise SEQ ID NO:1 the 28-619 residue amino acid sequence and have maximum 20 amino acid whose replacements, deletion or changes and oppositely do not influence the expression of SEQ ID NO:1 encoded protein or active polypeptide.
In another embodiment, the invention provides the DIG-109 toxin protein of separation, comprise and be selected from following active toxin fragment:
The polypeptide of amino acid sequence that comprises the 1-619 residue of SEQ ID NO:1;
Comprise the polypeptide that has with the amino acid sequence of the amino acid sequence at least 90% sequence homogeneity of the 1-619 residue of SEQ ID NO:1;
Comprise SEQ ID NO:1 the 1-619 residue amino acid sequence and have maximum 20 amino acid whose replacements, deletion or changes and oppositely do not influence the expression of SEQ ID NO:1 encoded protein or active polypeptide.
In another embodiment, the invention provides the plant that comprises DIG-109 albumen.
In another embodiment, the invention provides the method for Pest Control population, comprise said population is contacted with the DIG-109 albumen of insecticidal effective dose.
In another embodiment, the invention provides the nucleic acid of the separation of encoding D IG-109 albumen.
In another embodiment, the invention provides DNA construct, its nucleotide sequence that comprises encoding D IG-109 albumen may be operably coupled to promotor, and wherein said promotor is not derived from bacillus thuringiensis and can in plant, drives expression.The present invention also provides and has comprised this DNA construct stable integration and advance its genomic genetically modified plants, and protective plant avoids the method for insect pest, comprises this construct is introduced in the said plant.
The sequence general introduction
SEQ ID NO:1 Cry1Ca active toxin fragment; 619aa
SEQ ID NO:2 the one Cry1Ab parent toxin fragment; 545aa
SEQ ID NO:3 DIG-152 chimeric protein; 1164aa (Pf version)
SEQ ID NO:4 the 2nd Cry1Ab parent toxin fragment; 545aa
SEQ ID NO:5 DIG-109 chimeric protein; 1164aa (corn version)
SEQ ID NO:6 Cry1Ca436 polypeptide; 10aa
SEQ ID NO:7 Cry1Ca591 polypeptide; 10aa
The CDS of corn-optimization of SEQ ID NO:8 encoding D IG-109; 3492bp
SEQ ID NO:9 ZGP3S oligonucleotides; 21nt
SEQ ID NO:10 ZGP3A oligonucleotides; 21nt
SEQ ID NO:11 TQZGP3 oligonucleotides; 23nt
SEQ ID NO:12 DSM2S oligonucleotides; 17nt
SEQ ID NO:13 DSM2A oligonucleotides; 19nt
SEQ ID NO:14 DSM2FQ oligonucleotides; 20nt
SEQ ID NO:15 CRY1CaS oligonucleotides; 18nt
SEQ ID NO:16 CRY1CaA oligonucleotides; 18nt
SEQ ID NO:17 Cry1Ca oligonucleotides; 23nt
SEQ ID NO:18 AAD1S oligonucleotides; 20nt
SEQ ID NO:19 AAD1A oligonucleotides; 22nt
SEQ ID NO:20 AAD1 oligonucleotides; 24nt
SEQ ID NO:21 Y1CAS oligonucleotides; 18nt
SEQ ID NO:22 Y1CAR oligonucleotides; 18nt
SEQ ID NO:23 F6Y1CA oligonucleotides; 23nt
SEQ ID NO:24 IVF-Taq oligonucleotides; 18nt
SEQ ID NO:25 IVR-TAQ oligonucleotides; 19nt
SEQ ID NO:26 IV-Probe oligonucleotides; 26nt
SEQ?ID?NO:27?DIG-110；1079aa
The DIG-110 coding region of SEQ ID NO:28 corn-optimization; 3237bp
SEQ?ID?NO:29?DIG-111；543aa
The DIG-111 coding region of SEQ ID NO:30 corn-optimization; 1629bp
SEQ?ID?NO:31?DIG-112；1044aa
The DIG-112 coding region of SEQ ID NO:32 corn-optimization; 3132bp
SEQ?ID?NO:33?DIG-113；508aa
The DIG-113 coding region of SEQ ID NO:34 corn-optimization; 1524bp
SEQ?ID?NO:35?DIG-114；582aa
The DIG-114 coding region of SEQ ID NO:36 corn-optimization; 1746bp
Detailed Description Of The Invention
DIG-109 and DIG-152 albumen, and insecticidal activity variant.Except the DIG-152 albumen of the DIG-109 albumen of the SEQ ID NO:5 of total length and SEQ ID NO:3, the present invention comprises the insecticidal activity variant.For term " variant ", the applicant wants to comprise fragment, the mutant of some deletion or insertion, and some fusion.The Cry1Ca active toxin fragment of DIG-109 and DIG-152 is classical three domain C ry albumen (classic three-domain Cry protein).Be included in DIG-109 and the preface of DIG-152 protein variant in the present invention as description, be necessary to look back briefly common and the structure of three domain C ry albumen of DIG-109 and DIG-152 proteotoxin particularly.
Most bacillus thuringiensis δNei Dusu crystalline protein molecule is made up of two functional fragments.The protease resistant active toxin is first fragment and corresponding to about the first half of said protein molecular.The parent toxin molecule of the about 130kDa of total length is the resistance core fragment through the protease rapid processing in insect gut.The fragment of in this process, being deleted is called as " parent toxin fragment " in this article.The parent toxin fragment is considered to participate in the toxin crystal and forms (Arvidson et al., (1989)).Thereby the protease processing (Haider et al., (1986)) that the parent toxin fragment maybe be through reducing lps molecule or reduce toxin solubility (Aronson et al., (1991)) thus limit core to insect near passing on part insect specificity.B.t. toxin is even in certain type, also in its length and different on the accurate location that the parent toxin fragment changes from the active toxin fragment.The active toxin fragment to the transformation of parent toxin fragment typically occur in the toxin total length about 50% to about 60% between.SEQ ID NO:3 discloses 1164 amino acid whose sequences of DIG-152 polypeptide total length, and wherein 619 amino acid of N-end comprise the disclosed Cry1Ca active toxin with SEQ ID NO:1.SEQ ID NO:5 discloses 1164 amino acid whose sequences of DIG-109 polypeptide total length, and wherein 619 amino acid of N-end comprise the Cry1Ca active toxin.
Confirmed Cry1Aa1, Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, the three-dimensional crystalline structure of Cry4Ba and Cry8Ea1.The structure of these active toxins is closely similar and have hereinafter described the different structure territory of characteristic by three and form (summarizing the al. in de Maagd et, 2003).
Domain I is seven alpha-helix bundles, and wherein spiral 5 is surrounded by 6 both sexes spirals.This domain relates to the formation in hole and comprises that with other hole formation albumen hemolysin and colicine have autoploidy.The domain I of Cry1Ca active toxin albumen comprises the 36-254 amino acids residue of SEQ ID NO:1.[be to be understood that DIG-109 and DIG-152 chimeric protein comprise Cry1Ca active toxin fragment, thereby distribute to the amino acid sequence of disclosed DIG-152 chimeric protein among amino acid sequence and the SEQ ID NO:3 that equivalent like the amino acid sequence of disclosed Cry1Ca active toxin fragment among the SEQ ID NO:1 also is applicable to disclosed DIG-109 chimeric protein among the SEQ ID NO:5.]
Domain II is assembled in a β triangular prism by 3 antiparallels and forms.The ring of this domain is played an important role in combining the insect midgut acceptor.In Cry1A albumen, the surface that exposes ring on domain II βZhe Die top relates to and combines Lepidoptera cadherin acceptor.Cry3Aa domain II ring combines the film associated metal protease (Ochoa-Campuzano et al., 2007) of colorado potato beetle (Leptinotarsa decemlineata is also referred to as the Colorado colorado potato beetle) in a similar fashion.Domain II combines albumen to comprise that yolk and jacaline have autoploidy with some carbohydrate.Cry1Ca active toxin albumen comprises the 262-458 amino acids residue of SEQ ID NO:1.
Domain II I is the β sandwich structure of 2 antiparallels.For example the carbohydrate binding structural domain of dextranase, galactose oxidase, sialidase etc. is relevant for this domain and protein on the structure.Some type of domain II I bind receptor albumen also possibly participated in the insertion with the oligomerization toxin pronucleus of the receptor acting of second class, and the example is aminopeptidase and the alkaline phosphatase (Pigott and Ellar, 2007) under the situation of Cry1A albumen.Similarly Cry domain II I acceptor is also differentiated in coleoptera.Conservative B.t. sequence blocks 2 and 3 lays respectively near the N-end and C-end of domain II.Therefore, these conserved sequence pieces 2 and 3 are roughly borderline regions between 3 functional structure territories.The zone of these conserved dnas and albumen homology have been used to Development Engineering property reorganization B.t. toxin (US Patent No.6090931, WO 1991/01087, WO 1995/06730, WO 1998/022595).The domain II I of Cry1Ca albumen comprises the 468-617 amino acids residue of SEQ ID NO:1.
The alpha-helix 1 of having reported domain I is removed receptors bind afterwards.Aronson et al. (1999) has proved that the Cry1Ac that is bonded to BBMV is protected from the cut-out of Proteinase K from residue 59 beginnings, promptly just in time after alpha-helix 1; Similarly the result is also quoted by Cry1Ab.Gomez et al., (2002) find that the Cry1Ab oligomer that on the BBMV receptors bind, forms lacks alpha-helix 1 part of domain I.Equally; Soberon et al.; (2007) showed lacking of Cry1Ab and Cry1Ac comprise alpha-helix 1 on the Cry three-dimensional structure about 60 amino acid whose N-end deletion mutants can the set of monomers of the about 60kDa of molecular weight be put into before core, under the situation that the cadherin combination lacks.These N-end deletion mutants are in the news Cry resistant insects larva are had activity.Further, Diaz-Mendoza et al., the Cry1Ab fragment of 43kDa and 46kDa has been described in (2007), and it keeps the activity to Mediterranean corn borer (moth stem noctuid, Sesamia nonagrioides).These fragments are proved and comprise amino acid residue 116-423; Yet its accurate amino acid sequence is not illustrated, and the activity mechanism of these proteolytic fragments also is unknown.Gomez et al., (2002), Soberon et al.; 2007 with Diaz-Mendoza et al.; (2007) result and Hofte et al., the formation contrast of (1986), the latter has reported that 36 amino acid of N-end of disappearance Cry1Ab can cause losing of insecticidal activity.
Because its structure is known, through the amino acid sequence of Cry1Ca amino acid sequence and Cry8Ea1 relatively, we have reasoned out the spiral 1 among the domain I of Cry1Ca active toxin; 2A; 2B, 3 and 4 starting and ending point, and the position in the space between them (spacer) zone.These location expressions are in table 1.
The amino acid coordinate of the alpha-helix of the plan of table 1.Cry1Ca active toxin albumen.
Figure BDA00002019672300061
The amino terminal deletion mutant of DIG-109 and DIG-152.The invention provides the mutant of DIG-109 and DIG-152 in one aspect, wherein alpha-helix 1, and all or part of of 2A and 2B deleted to improve insecticidal activity and to avoid insect development resistance.These changes are used to provide DIG-109 and the mutant of DIG-152, for example improved targeted insect type spectrum, potentiality and the insect-resistant management with improved performance.In embodiments more of the present invention, the change of object possibly influence the active effect of parent toxin and core forms, and causes insect to poison.More particularly, in order DIG-109 with improved performance and the mutant of DIG-152 to be provided, to have described the part of progressively deleting with the gene of removing coding N-end.Whole and alpha-helix 2 all or part of of alpha-helix 1 among the domain I removed in this deletion, keeps the structural intergrity of alpha-helix 3 to 7 simultaneously.Thereby this invention scheme part relates to alpha-helix component through operating structure territory I to improve Cry albumen effect, for more effective core forms.More particularly, the scheme of this invention partly relates to improved DIG-109 and DIG-152 albumen, and it is designed to have N-end disappearance, and this zone has the autoploidy of alpha-helix 1 and 2 secondary structures among that infer and domain I Cry1 albumen.
For the deletion of the insecticidal properties that improves DIG-109 and DIG-152 toxin can begin before the alpha-helix 2A starting point of prediction, and can after the end of alpha-helix 2B, finish, still preferably not extend to alpha-helix 3.
When the coded sequence of design N-end deletion mutant, the ATG initiation codon of a coding methionine is inserted 5 ' end of the nucleotide sequence that is designed to express said deletion mutant.For the sequence that is designed in the genetically modified plants, preferably come bonding according to Varshavsky (1997) " the N-hold-carrying then ".Its some amino acid of instruction can contribute for lability and the degraded of albumen in eukaryotic when its N-as protein holds residue.For example, it is F that the data show N-that is collected by yeast and mammalian cell holds unstable amino acid residue, L, W, Y, R, K, H, I, N, Q, D, E and possible P.Although the degradation mechanism of specific protein can be different between species, N-mentioned above holds the conservative of unstable amino acid whose homogeneity to hint that similar mechanism possibly work in plant cell.For example, Worley et al., (1998) find that the N-hold-carrying then comprises alkalescence and aromatic residues in plant.The proteolysis of plant rennet cuts off and can expose unstable n terminal amino acid near the starting point of the alpha-helix 3 of B.t. insecticidal proteins object probably.The quick decay of the protein that such process can cause cutting off and restriction B.t. insecticidal proteins build up to the level that is enough to effectively control insect.Thereby for by the initial N-end deletion mutant of unstable amino acid, the applicant preferably increases between the methionine of translation initiation and unstable amino acid and specifies the amino acid whose codon of G (glycine).
Embodiment 13 and 14 has provided the particular instance according to the aminoterminal deletion mutant of DIG-109 of the present invention and DIG-152.Other useful fragment can be differentiated through the insect bioassay of the fragment of trypsase or pancreas milk reducing protease digesting acquisition through total length soluble crystal albumen; To confirm that which fragment keeps toxicity, perhaps can differentiate through definite toxalbumin fragment of encoding by the dna fragmentation in Cry encoding histone zone.This albumen will mainly have short N-end and long C-end blocks, than parent toxin.The N end of minimum toxin fragment is confirmed through the n terminal amino acid sequencing of the soluble crystal albumen of trypsase or chymotrypsin processing through technology known in the art.
Chimeric toxin.The chimeric protein of having reported utilizes the active toxin of a Cry toxin and the parent toxin fragment of another Cry toxin to merge.DIG-109 and DIG-152 variant comprise such toxin; Its N-end endotoxin core fragment (it can be total length or have N-end disappearance mentioned above) that comprises a Cry1Ca toxin is blended in the parent toxin fragment of an xenogenesis, on certain site after this active toxin fragment end.Transformation to said xenogenesis parent toxin fragment can occur in about active toxin/parent toxin binding site, and perhaps alternatively, the part (extending beyond the active toxin fragment) of self parent toxin can be retained, and then occurs in downstream to the transformation of xenogenesis parent toxin.For example, chimeric toxin of the present invention has total length active toxin fragment (amino acid/11-619) and the xenogenesis parent toxin (amino acid 620-C is terminal) of Cry1Ca.In preferred implementation, the xenogenesis fragment of parent toxin is derived from the Cry1Ab δNei Dusu, shown in SEQ ID NO:2 and SEQ ID NO:4.
protease susceptibility variant.Insect gut protease typically reacts on and helps the insect amino acid that acquisition needs from food proteins.Studying the most thorough insect digestible protein enzyme is serine protease, and it (Englemann and Geraerts, 1980) occur with modal type, especially in the Lepidoptera species.Coleopteron has more neutral to acid enteron aisle than Lepidoptera enteron aisle.The main member of coleoptera larvae and adult, for example the Colorado colorado potato beetle has the subacidity midgut, and cysteine proteinase provides main proteolytic activity (Wolfson and Murdock, 1990).More accurately, Thie separates with Houseman (1990) and has characterized the cysteine proteinase in the colorado potato beetle of Colorado, cathepsin B's appearance and Cathepsin H's appearance and asparagine protease, cathepsin D's appearance.Gillikin et al., (1992) have characterized the proteolytic activity in the western corn rootworm enteron aisle, and have found main cysteine proteinase.The US patent No. 7230167 discloses serine protease, and cathepsin G is present in the western corn rootworm.The diversity of insect gut protease and different activities can influence the susceptibility of insect to specific B.t. toxin.
In another embodiment of the invention, protease cutting site can be processed on definite site with the protein in the midgut of the susceptible larva that influences specific insect pest and handle (Walters et al., 2008).These protease cutting sites can be introduced because of synthetic or the overlapping PCR of montage methods such as (Horton et al., 1989) through for example chemical based.For example, the serine stretch protein enzyme recognition sequence, the specific site that can insert the Cry protein structure alternatively is with the processing of albumen in the midgut that influences the susceptible larva on the deletion site of confirming.Can comprise Lepidoptera midgut serine protease for example trypsase or trypsin-like enzyme by the serine protease that this mode is developed, chymotrypsin, elastoser, or the like (Christeller et al., 1992).Further, perhaps the deletion site of empirical evaluation can be processed to influence protein active through being bonded to the BBM carrier to handle the order-checking of the Cry proteopepsis product that produces through classification larva midgut proteinase not.Have the improved activity of lepidoptera pest through gene elmination or through the Cry albumen of introducing the change that protease cutting site produces; Comprise European corn borer (Ostrinia nubilalis), southwestern corn straw crambid (Diatraea grandiosella), cotton bollworm (Helicoverpa zea); Black cutworm (Agrotis ipsilon); Fall army worm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), little sugarcane borer (Diatraea saccharalis); Loxagrotis albicosta and other target pest.
The coleoptera serine protease is trypsase for example, chymotrypsin and cathepsin G's appearance protease, and the coleoptera cysteine proteinase is cathepsin (B appearance for example; L appearance, O appearance and K appearance protease) (Koiwa et al.; (2000) and Bown et al., (2004)), the coleoptera metalloproteinases is ADAM10 (Ochoa-Campuzano et al. for example; (2007)) and coleoptera aspartic protease for example cathepsin D's appearance and E appearance, pepsin; Plasmepsin and rennin can be further through processing suitable recognition sequence to influence the processing of Cry albumen in the midgut of the susceptible larva of specific insect pest in the treatment site of confirming.
An optimum position introducing such protease cutting site is " space " zone between alpha-helix 2B and the alpha-helix 3, for example within the amino acid 85-90 of Cry1Ca active toxin albumen (SEQ ID NO:1 and table 1).Have the improved activity of insect pest through gene delection or through introducing the Cry albumen that protease cutting site changes, include but not limited to the autumn mythimna separata, sugarcane moth borer or the like.
The amino acid whose sequence of N-end that exists multiple technologies to measure to comprise polypeptide or C-end residue.For example, the automatic edman degradation methodology can be used for sequence measurement to measure n terminal amino acid sequence to 30 amino acid residue and to have the accuracy of each residue 98%.Further, the amino acid whose sequence of measuring the c-terminus comprise polypeptide also is possible (Bailey et al., (1992); US Patent No.6046053).Therefore, in some embodiments, by the method through proteolytic treatment, for example, through the protease of insect gut preparation, the B.t.Cry albumen that is activated can be characterized, and differentiates the N-end or the C terminal amino acid of the toxin fragment of said activation.Through introduce or eliminate at the correct position of coded sequence the Protease Treatment site with allow or eliminate insect, plant or microbial protease bigger misfolded proteins the proteolysis cutting DIG-109 and DIG-152 variant also within the scope of the invention.The final result of such operation is understood that it is to produce to have active toxin sheet segment molecule identical with complete (total length) toxin protein or better activity.
The domain of DIG-109 and DIG-152 toxin.The separated structures territory of the Cry1Ca active toxin fragment of in DIG-109 and DIG-152 toxin, giving an example; The domain that (and with these domains the variant of 90%, 95% or 97% homogeneity is arranged) is supposed to be used for from other Cry toxin combines the new toxin with the protein stability that the insect toxicity spectral pattern with increase, improved potentiality or increase are provided.The domain I of Cry1Ca active toxin albumen is made up of the 36-254 amino acids of SEQ ID NO:1.The domain II of Cry1Ca active toxin albumen is made up of the 262-458 amino acids of SEQ ID NO:1.The domain II I of Cry1Ca active toxin albumen is made up of the 468-617 amino acids of SEQ ID NO:1.Domain exchanges or moves is the mechanism that produces variable delta-endotoxin proteins.Domain II and III can exchange between delta-endotoxin proteins, cause producing heterozygosis or chimeric toxin with improved insecticidal activity or target spectrum.Domain II relates to receptors bind.Domain II I combines the receptor protein of some type and maybe be before the oligomerization toxin works in the insertion of core.Some domain II I substituents in other toxin have demonstrated super-active (the de Maagd et al. that produces to beet armyworm (Spodoptera exigua); And have an enlightenment (Knight et al., (2004)) of designs C ry toxin structure territory exchange (1996)).
Produce recombinant protein and the method that detects its insecticidal activity and be well known in the art (referring to, for example, Naimov et al., (2001); De Maagd et al., (1996), Ge et al., (1991); Schnepf et al., (1990), Rang et al., (1999)).After deliberation Cry1A and Cry3A albumen domain I insertion and in film, form the ability in hole.The alpha-helix 4 of domain I and 5 inserts and the hole plays key effect (Walters et al., 1993, Gazit et al., 1998 in forming at film; Nunez-Valdez et al., 2001), and other spiral is inferred the surface of contact membranes, just as the rib of umbrella such (Bravo et al., (2007); Gazit et al., (1998)).
are through few amino acids disappearance, replacement or add DIG-109 and the DIG-152 variant of creating.The amino acid deletions of the amino acid sequence of the Cry1Ca active toxin fragment of SEQ ID NO:1, replacement or interpolation can easily be carried out through the sequence mode, and the effect of the insecticidal activity of these mutant can detect through bioanalysis.If the number that changes is a minority, such detection can not comprise excessive experiment.The present invention includes the insecticidal activity variant of active toxin (the 1-619 amino acids of SEQ ID NO:1), wherein have maximum 10, maximum 15, or maximum 20 independently amino acid whose interpolations, disappearance or replacement.
The present invention includes DIG-109 and DIG-152 variant, it has and the 1-619 amino acids 90%, 95% of SEQ ID NO:1 or the active toxin fragment of 97% homogeneity.Variant can produce through random mutation, perhaps can design variable.Under the situation of the sudden change that designs; Having very, high likelihood produces the variant that has with the similar activity of body toxin; If the important area at toxin keeps amino acid homogeneity; Said important area works to BA or relates to the definite of three-dimensional conformation, and it finally also is the biologically active service.Amino acid can be arranged in following classification: nonpolar; Uncharged polar, alkalescence, and acid.Conservative replaces, and promptly wherein one type amino acid replaces with another amino acid of same-type, has the minimum possibility of the BA that changes said variant basically.Table 2 provides the tabulation of the amino acid whose instance that belongs to each type.
Table 2.
Figure BDA00002019672300111
In some instances, also can carry out non-conservative replacement.Material facts are that these replace the BA that should not damage toxin significantly.Variant comprises owing to sudden change polypeptide different on amino acid sequence.The misfolded proteins that the present invention comprises is a BA, and promptly they continue to have the BA of this body protein,, keeps insecticidal activity that is.
But misfolded proteins also can be designed as difference on the sequence level has kept identical or similar basic generally three-dimensional structure, and surface charge distributes, or the like.Referring to the for example US patent No. 7058515; Larson et al., (2002); Stemmer (1994a, 1994b, 1995); With Crameri et al., (1996a, 1996b, 1997).
nucleic acid.The nucleic acid of the separation of encoding D IG-109 toxin or encoding D IG152 toxin is one aspect of the present invention.This comprises coding SEQ ID NO:1, the nucleic acid of SEQ ID NO:3 and SEQ ID NO:5, and complement, and coding SEQ ID NO:1, other nucleic acid of the desinsection variant of SEQ ID NO:3 and SEQ ID NO:5.Meaning this nucleic acid molecules with " separation " applicant shifts out and is placed in the different environment with staff from its original environment.The disclosed amino acid sequence of this paper because the redundancy of genetic codon, multiple different dna sequence dna can be encoded.Those skilled in the art are easy to set up the optional dna sequence dna of the same or same basically toxin of coding.
gene is synthetic.The gene of improved Cry albumen described herein of encoding can be made through multiple method well known in the art.For example, synthetic gene fragment and synthetic gene can be made (Caruthers et al, 1987) through tris phosphite and phosphorous acid amination, and it is synthetic to allow carrying out gene according to demand through commercial supplier.Full-length gene can assemble through number of ways, for example comprises, through connecting the overlapping oligonucleotides (Stewart and Burgin, 2005) of restriction fragment or PCR assembling.Further, the terminal gene disappearance can realize through the pcr amplification that uses the terminal oligonucleotides of locus specificity.
The nucleic acid of encoding D IG-109 toxin or DIG-152 toxin can come synthetic property structure through any one method that provides of for example a plurality of commercial supplier.(referring to for example, US Patent No.7482119B2).These genes, or its part or variant, also can through for example use gene synthetic with appointed method for example US Patent No.5380831 come synthetic property structure.Alternatively, the variant of gene synthetic or that exist naturally can make up with the standard molecular biological technique of manufacturing place sudden change easily.The fragment of these genes also can prepare according to standard procedure with the exonuclease or the endonuclease of commercial permission.For example, for example Bal3I or some orthomutation can be used for the cut-out nucleotide from the end system ground of these genes to enzyme.In addition, the genetic fragment of coding active toxin fragment can obtain with multiple Restriction Enzyme.
The amino acid sequence of known DIG-109 toxin or DIG-152 toxin; Can design coded sequence through following method; Promptly, use optional (redundancy) codon to come Orders Corrected then to remove the sequence that possibly cause problem with counter this protein sequence of translating of the preferred codon of target host.Further, the amortization codon can be processed into non-coding and read frame, unintentionally ORFs long to eliminate.
are sequence homogeneity quantitatively.In order to measure the percentage homogeneity of two amino acid sequences or two nucleotide sequences, this sequence is compared with the best comparison purpose.Percentage homogeneity between two sequences is the function (that is, percentage homogeneity=identical bits is counted/total number of sites (for example, overlapping site) X100) of the number of the total same loci of sequence.In an embodiment, these two sequences are same length.Percentage homogeneity between two sequences can be confirmed with the similar techniques that hereinafter is described, comprise or do not comprise allowable clearance.When calculated percentage homogeneity, typically calculate accurately pairing.
The confirming of percentage homogeneity between two sequences can use mathematical algorithm to accomplish.A limiting examples of this algorithm is BLAST (Altschul et al., 1990 and Karlin and Altschul, 1990), is revised by Karlin and Altschul (1993), and incorporates BLASTN and BLASTX program into.Blast search can be advantageously used in nucleic acid or Protein Data Bank differentiating the sequence with search sequence homology (similar).The BLASTN search can be carried out, and (score=100, word length=12) are used to differentiate that the nucleic acid molecules with requirement of the present invention has the nucleotide sequence of autoploidy.The BLASTX search can be carried out, and (score=50, word length=3) are used to differentiate the amino acid sequence that has autoploidy with the insecticidal proteins molecule of requirement of the present invention.
Gap BLAST (Altschul et al., (1997)) can be used to obtain the gap comparison of comparison purpose.Alternatively, PSI-Blast can be used to carry out repeat search, and it surveys relation (Altschul et al., (ibid.)) far away between two molecules.When using BLAST, when Gapped BLAST and PSI-Blast program, can use the default parameters of program separately.Referring to www.ncbi.nlm.nih.gov.
A limiting examples that is used for the mathematical algorithm of comparative sequences is ClustalW algorithm (Thompson et al., 1994).The ClustalW comparative sequences is also compared amino acid or the integral body of dna sequence dna, thereby the data about the sequence conservation of whole amino acid sequence or nucleotide sequence can be provided.The ClustalW algorithm is used for the multiple commercial DNA/ amino acid analysis software kit that allows, for example Vector NTI Program Suite (Invitrogen, Inc., Carlsbad, ALIGNX module CA).When with ALIGNX comparison amino acid sequence; Can use default setting easily; Comprise the open point penalty in gap 10 minutes, gap extension point penalty 0.1 minute and blosum63mt2 comparator matrix are to assess two percentage amino acid similarity (uniformity) or homogeneity between the sequence.When with ALIGNX comparison dna sequence dna, can use default setting easily, comprise the open point penalty in gap 15 minutes, gap extension point penalty 6.6 minutes and swgapdnamt comparator matrix are to assess two percentage homogeneity between the sequence.
Another limiting examples that is used for the mathematical algorithm of sequence contrast is Myers and Miller (1988).Such algorithm is merged in the wSTRETCHER program, and this is the part (allowing to obtain from http://emboss.sourceforge.net/) of wEMBOSS sequence alignment software kit.WSTRETCHER uses the modification algorithm of the classical dynamic routine algorithm of linear space to calculate the optimum whole world comparison of two sequences.Can specify the replacement matrix, the gap that are used to calculate comparison to insert point penalty and gap extension point penalty.When using the wSTRETCHER program to come the comparison nucleotide sequence, can use open point penalty in gap 16 minutes and gap extend point penalty 4 minutes and sub matrix file EDNAFULL.When being used for the comparing amino acid sequence, can use open point penalty in gap 12 minutes and gap extend point penalty 2 minutes and sub matrix file EBLOSUM62.
The further limiting examples that is used to contrast the mathematical algorithm of sequence is Needleman and Wunsch (1970), and it incorporates sequence alignment software kit GAP Version 10 and wNEEDLE (http://emboss.sourceforge.net/) into.GAP Version 10 can be used for confirming sequence homogeneity or similitude, uses following parameters: for nucleotide sequence, % homogeneity and % similitude use GAP Weight be 50 and Length Weight be 3 and nwsgapdna.cmp get sub matrix.For amino acid sequence, % homogeneity and % similitude use GAP Weight be 8 with Length Weight be 2 and BLOSUM62 get sub matrix.
WNEEDLE reads two list entries, finds the optimum comparison (comprising the gap) along whole sequence, and writes out its optimum global sequence alignment and submission.This algorithm uses all possible comparison and selects preferably, uses the matrix comprise the right numerical value of each possible residue or oligonucleotide ligand.WNEEDLE finds the comparison of the score with maximum possible, the score of one of them comparison equal from the summation of pairing of sub matrix, deduct in aligned sequences open and extend the point penalty that the gap obtains.Substitution matrix and gap are opened and are extended point penalty and can the user specify.When the comparing amino acid sequence, the open point penalty in acquiescence gap is 10 minutes, and it is 0.5 minute that point penalty is extended in the gap, and uses the EBLOSUM62 comparator matrix.When using wNEEDLE comparison dna sequence, the open point penalty in acquiescence gap is 10 minutes, and it is 0.5 minute that point penalty is extended in the gap, and uses the EDNAFULL comparator matrix.
Also can use equivalent program." equivalent program " is meant any sequence contrast program; It is to any two object sequence; Generation has comparison and the same percentage sequence homogeneity of identical nucleotide or amino acid residue pairing, than by ALIGNX, and the comparison of the correspondence of wNEEDLE or wSTRETCHER generation.% homogeneity be between two sequences identical pairing relatively the comparison zone of report percentage (being included in any gap on the length) and the % similitude is two pairings between sequence percentages (being included in any gap on the length) in the comparison zone of report relatively.
Comparison also can be carried out through hand inspection.
recombinant host.Toxin encoding gene of the present invention can be introduced multiple microorganism or plant host.The expression of toxin gene directly or indirectly causes producing in the cell and keeping insecticidal proteins.For suitable microbial hosts, pseudomonad for example, microorganism can be provided in the environment of insect, and wherein they will be bred and be ingested.Consequently controlled insect.Alternatively, the microorganism that contains toxin gene can be handled under certain condition, and this condition can prolong toxin activity and stabilized cell.The cell of handling, it is active that it keeps toxin, can be provided in the environment of target pest subsequently.
When the B.t. toxin gene is introduced microbial hosts through suitable carriers, and said host is provided in environment with animation, is necessary to use specific microorganism.Selection is considered to occupy the microbial hosts of " phytosphere phytosphere " (blade face phylloplane, leaf circle phyllosphere, root circle rhizosphere, and/or root face rhizoplane) of one or more interested crop.Select these microorganisms winning in particular environment (crop and other insect residence) and the competition of wild native country microorganism; Gene with coded polypeptide insecticide that stable maintenance and expression are provided; And, desirably, the improvement protection that provides insecticide to avoid environment degradable and inactivation.
Known a large amount of microorganism is present in blade face (surface of leaves of plants) and/or the root circle (soil around the plant roots) of multiple important crop.These microorganisms comprise bacterium, algae, and fungi.Attracting especially is microorganism, and bacterium for example is for example with the subordinate: pseudomonas Pseudomonas, Erwinia Erwinia; Serratia Serratia, Klebsiella Klebsiella, Xanthomonas Xanthomonas; Streptomyces Streptomyces, rhizobium Rhizobium, Chinese rhizobium Sinorhizobium; Rhodopseudomonas Rhodopseudomonas, Methylophilius, Agrobacterium Agrobacterium; Acetic acid Pseudomonas Acetobacter, genus lactubacillus Lactobacillus, Arthrobacter Arthrobacter; Azotobacter Azotobacter, Leuconostoc Leuconostoc and Alcaligenes Alcaligenes; Fungi, especially yeast, for example descend dependent of dead military hero: saccharomyces cerevisiae belongs to Saccharomyces; Cryptococcus Cryptococcus, Kluyveromyces Kluyveromyces, Sporobolomyces Sporobolomyces; Rhodotorula Rhodotorula and Aureobasidium Aureobasidium.Interested especially is for example pseudomonas syringae Pseudomonas syringae of these phytosphere bacterium kinds; Pseudomonas fluorescens Pseudomonas fluorescens; Serratia marcesens Serratia marcescens, pyroligneous acid bacterium Acetobacter xylinum, agrobacterium tumefaciens Agrobacterium tumefaciens; Radioactivity Agrobacterium Agrobacterium radiobacter; Rhodopseudomonas spheroides Rhodopseudomonas spheroides, xanthomonas campestris Xanthomonas campestris, Sinorhizobium meliloti Sinorhizobium meliloti (rhizobium melioti Rhizobium meliloti originally); Alcaligenes eutrophus Alcaligenes eutrophus and azotobacter vinelandii Azotobacter vinelandii; With phytosphere yeast kind rhodothece rubra Rhodotorula rubra for example, rhodotorula glutinis R.glutinis, herbage rhodotorula R.marina; Orange red yeast R.aurantiaca, light white latent ball yeast Cryptococcus albidus, C.diffluens; Cryptococcus laurentii C.laurentii, Luo Shi yeast Saccharomyces rosei has spore torula S.pretoriensis; Saccharomyces Cerevisiae in S .cerevisiae, shadow yeast Sporobolomyces roseus, fragrance shadow yeast S.odorus; Kluyveromyces veronae and Aureobasidium pollulans.Interested especially is coloured microorganism.
The method of control insect pest
When insect contacts the toxin of the effective dose of sending through genetically modified plants expression, the protein compositions of filling a prescription, spraying protein compositions, bait matrix or other delivery system; The result is the significant death of insect; Perhaps insect its source of no longer taking food, this makes toxin inapplicable to insect.
The object archon can be through number of ways " application " or be provided in the contact target insect.For example, can use genetically modified plants (wherein albumen is produced by plant and is present in the plant), this is well known in the art.The expression of toxin gene also can realize optionally in the particular organization of plant, root for example, and leaf, or the like.This can tissue-specific promoter realizes through for example using.Spray application is another instance and also is well known in the art.Object albumen can be as required final use and prescription easily; Spray then (or alternate manner provides) on the plant that will protect and/or around the plant/or plant near---before insect comes to light; After targeted insect comes to light, before finding and after finding, or the like.For example, the bait particle also can use and is well known in the art.
Genetically modified plants
Object protein can be used to protect the plant of any kind almost to avoid the injury of lepidopterous insects.The instance of such plant comprises corn, sunflower, and soybean, cotton, rape, paddy rice, Chinese sorghum, tobacco, wheat, barley, vegetables, ornamental plants, pepper (comprising capsicum), beet, fruit and turf are except sub-fraction.The method that transforms plant is well known in the art, and illustrative method for transformation is described among the embodiment.
A preferred implementation of the present invention is the gene-transformed plant with coded object insecticidal proteins or its variant.Because in the cell of plant transformed, have object insecticidal proteins or its variant of controlled quentity controlled variable, plant transformed has resistance to the invasion and attack of insect target pest.The genetic stocks of the insecticidal properties through the B.t. Pesticidal toxins of will encoding is integrated into Plant Genome, and said plant is eaten by specific insect pest, and then adult or larva will be dead after the consumed foods plant.The multiple member of monocotyledon and dicotyledon classification can be transformed.Transgenic crop and fruits and vegetables have commercial appeal.Such crop includes but not limited to corn, paddy rice, and soybean, rape, sunflower, clover, Chinese sorghum, wheat, cotton, peanut, tomato, potato, or the like.Exist multiple technologies with exogenic heredity material introduced plant cell, and obtain the plant that stable maintenance is also expressed the gene of being introduced.These technology comprise that genetic stocks encapsulates to the microparticle after-acceleration and directly get into cell (the US patent No. 4945050 with the US patent No. 5141131).Can use Agrobacterium technical transform plant, referring to the US patent No. 5177010, the US patent No. 5104310, european patent application No.0131624B1; European patent application No.120516, european patent application No.159418B1, european patent application No.176112, the US patent No. 5149645; The US patent No. 5469976, the US patent No. 5464763, the US patent No. 4940838, the US patent No. 4693976; European patent application No.116718, european patent application No.290799, european patent application No.320500, european patent application No.604662; European patent application No.627752, european patent application No.0267159, european patent application No.0292435, the US patent No. 5231019; The US patent No. 5463174, the US patent No. 4762785, the US patent No. 5004863 and the US patent No. 5159135.Other transformation technology comprises WHISKERS TMTechnology is referring to the US patent No. 5302523 and the US patent No. 5464765.Electroporation technology also is used to transform plant, referring to WO 1987/06614, and the US patent No. 5472869, the US patent No. 5384253, WO 1992/09696 and WO 1993/21335.All these transform patent and openly incorporate this paper into as a reference.Except the multiple technologies that transform plant, with the type of the tissue of foreign gene contact also can be different.Such tissue can include but not limited to that the embryo organizes, callus type I and II, and plumular axis, meristem, or the like.Nearly all plant tissue can use between the anti-idiophase that suitable technique transforms in those skilled in the art's scope.
The gene of encoding D IG-109 and DIG-152 toxin or its variant can insert plant cell like the disclosed multiple technologies well known in the art of preceding text through using.For example, comprise and be used for allowing to screen the label of microorganism transformed cell and have a large amount of cloning vectors of the dubbing system of function can be used for preparation and revise foreign gene Escherichia coli to insert higher plant.Such operation can comprise, for example, inserts sudden change, blocks, and adds, or replaces, and purposes is as required carried out.Said carrier comprises, for example, pBR322, pUC series, M13mp series, pACYC184, or the like.Thereby the sequence of coding Cry albumen or its variant can be inserted the suitable restriction site in the carrier.The plasmid that obtains is used for transformed into escherichia coli, and its cell is cultivated under suitable nutrient medium, gathers in the crops then and dissolves the plasmid that obtains available quantity with recovery.Sequence analysis, the restriction fragment analysis, electrophoresis and other biochemistry-molecular biology method carry out as analytical method routinely.After each operation, can cut off dna sequence dna and add next dna sequence dna.The dna sequence dna of each operation can be cloned in identical or other plasmid.
Containing the conversion that the T-DNA carrier is used for plant cell has obtained further investigation and has been described in european patent application No.120516 fully; Lee and Gelvin (2008), Fraley et al., (1986) and An et al., (1985), and set up perfect in this field.
Be be integrated into Plant Genome in case insert DNA, it will be relatively stable in the offspring.The carrier that is used for transformed plant cells contains one usually selects the label gene, and its coding makes institute's plant transformed cell have the albumen of resistance to a kind of weed killer herbicide or antibiosis, bialaphos for example, and kanamycin, G418, bleomycin, or hygromycin, or the like.Thereby the selection label gene that individuality has can allow to screen cell transformed, and the growth that does not contain the cell that inserts gene will be suppressed by screening compounds.
Multiple technologies can be used for DNA is inserted host plant cell.These technology comprise uses the T-DNA that sends as conversion reagent through agrobacterium tumefaciens or agrobacterium rhizogenes to transform.In addition, phytoplasm and the fusion that comprises the liposome of the DNA that need send, the direct injection of DNA, biological rifle transforms (microparticle bombardment), or electroporation, and other possible method, can use.
In a preferred implementation of the present invention, plant is transformed with gene, and the codon in encoding histone zone uses and optimized to the plant direction in the wherein said gene.Referring to, for example, the US patent No. 5380831, it is incorporated into as a reference at this.In addition, easily, can use the plant of the toxin of coding brachymemma.Typically encode about 55% to about 80% total length toxin of the toxin of brachymemma.The method that foundation is used for the synthetic property B.t. gene of plant is (Stewart 2007) known in the art.
Be not limited to transformation technology, gene preferably is integrated into the shuttle vector that is adapted at expressing in the plant cell B.t. insecticidal toxin gene or its variant, comprises plant promoter in this carrier.Except plant promoter, also can be effective to expression alien gene in the plant cell from the promotor in multiple source.For example, can use the promotor of bacterial origin, octopine synthase promoter for example, rouge alkali synthetase promoter, mannopine synthase promoter; The promotor in plant virus source, for example 35S of cauliflower mosaic virus and 19S promotor, or the like.Plant promoter includes, but are not limited to ribulose-1,5-bisphosphate, 6-diphosphonic acid (RUBP) carboxylase small subunit (ssu); The beta-conglycinin promotor; The phaseolin promotor, ADH (alcohol dehydrogenase) promotor, heat-inducible promoter; ADF (actin depolymerizing factor) promotor, and tissue-specific promoter.Promotor also can comprise some enhancer sequence element, and efficient is transcribed in its promotion.Typical enhancer includes but not limited to ADH1-introne 1 and ADH1-intron 6.Can use the constitutive character promotor.The constitutive character promotor guides the gene expression (for example, actin, ubiquitin, CaMV 35S) that continues in nearly all cell type with in nearly all time.Tissue-specific promoter forms gene expression in specific cell or tissue, for example leaf and seed (for example, zein, grease albumen, rape seed storage protein (napin), ACP (acyl group running albumen)) can use these promotors.Also can use promoters active during some stage of development of plants process and in specified plant tissue and organ.The instance of such promotor includes but not limited to promotor of root-specific, pollen specific, plumule specificity, corn silk specificity, cotton fiber specific property, seed endosperm specific property, phloem specific or the like.
Under particular environment, can use suitable inducible promoter.Thereby inducible promoter response signal specific also causes expression, said signal for example: physical stimulation (for example heat shock gene); Light (for example RUBP carboxylase), hormone (for example glucocorticoid); Antibiotic (for example tetracycline); Metabolite; And pressure (for example arid).Can also use the element of transcribing and translate of other needs that in plant, play function for example 5 ' not translate targeting sequencing, the rna transcription terminator sequence with gather adenosine and add burst.Various plants specific gene shuttle vector is known in the art.
The genetically modified crops that contain pest-resistant (IR) characteristic are very general and spread all over the North America in plants such as corn and cotton, and the use of these characteristics is just in global expansion.Many tame seeds companys have developed the commercialization genetically modified crops of combination IR and herbicide-resistant (HT) characteristic.This comprises the IR characteristic of giving through the B.t. insecticidal proteins and the combination of HT characteristic, and said HT characteristic for example tolerates for example sulfonylurea to acetolactate synthestase (ALS) inhibitor, imidazolone, and triazolo pyrimidine, sulfonanilide, or the like; Glutamine synthelase (GS) inhibitor such as bialaphos, careless ammonium phosphine, or the like; 4-medical midbodies of para (ortho)-hydroxybenzoic acetone acid dioxygenase (HPPD) inhibitor such as mesotrione, isoxaflutole, or the like; 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS) inhibitor such as glyphosate or the like; And acetyl-CoA carboxylase (ACCase) inhibitor such as cover grass energy, diclofop-methyl, Quizalotop-ethyl or the like.The albumen that transgenosis provides in other known instance provides the tolerance to weed killer herbicide to plant; The chemical species of said weed killer herbicide is Phenoxy acid herbicides and pyridine fluoroacetic acid growth hormone weed killer herbicide (referring to WO 2007/053482A2) for example; Or Phenoxy acid herbicides and fragrant phenoxy propionic ester weed killer herbicide are (referring to WO 2005107437A2, A3).Ability through IR Characteristics Control various pests problem is a kind of notion of commercially valuable, and the convenience of this product will be enhanced, and is combined in the same plant like fruit insect control characteristic and weeds control characteristic.In addition, for example preceding text are mentioned with one or more extra HT characteristic for the IR characteristic that can provide through independent plant combination B.t. insecticidal proteins scheme for example of the present invention, and add one or more extra input characteristics (insect-resistant that for example, is provided by B.t. extension or other insecticidal proteins; As the insect-resistant that mechanism such as RNAi provide, the nematode resistance that B.t. extension or other nematicide albumen provide is as the nematode resistance that mechanism such as RNAi provide, disease resistance; Force resistance, improved nutritional utilization, or the like); Perhaps output characteristics (for example, high oil content, healthy oil component; Nutrition improves, or the like), thereby obtain further to be worth.Such combination can add through traditional raising (raising heap, breeding stack) or as new transformation event, introduces when comprising several genes (molecule heap).Benefit comprises can manage insect pest and promote weeds control and provide to the producer and/or consumer's second benefit in crop plants.Thereby the present invention also can be used for the complete agronomy bag that has improved crop quality to provide of combining with other characteristic, and it can control any amount of agronomy incident with cost neatly effectively.
Target pest
DIG-109 toxin of the present invention and DIG-152 toxin are particularly suitable for controlling lepidopterous insects.Lepidoptera is one type of important agricultural, gardening and house pest, causes injury very in a large number its every year.This insect order comprises leaf-and the larva and the adult of root-feed.Lepidopterous insects includes, but are not limited to: Achoroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon (black cutworm); Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria; Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori; Bucculatrix thurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica; Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata; Diaphania nitidalis, Diatraea grandiosella (southwestern corn borer), Diatraea saccharalis (sugarcane borer), Ennomos subsignaria, Eoreuma loftini, Esphestia elutella; Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea; Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea (cotton bollworm); Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria; Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxagrotis albicosta (west beans noctuid), Loxostege sticticalis, Lymantria dispar; Macalla thyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata; Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.; Ostrinia nubilalis (European corn borer), Paleacrita vernata, Papiapema nebris (common borer), Papilio cresphontes, Pectinophora gossypiella; Phryganidia californica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypena scabra; Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella (diamond-back moth); Pontia protodice, Pseudaletia unipuncta (armyworm), Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna; Sitotroga cerealella, Spilonta ocellana, Spodoptera frugiperda (autumn mythimna separata), Spodoptera exigua (beet armyworm), Thaurnstopoea pityocampa; Ensola bisselliella, Trichoplusiani, Udea rubigalis, Xylomyges curiails and Yponomeuta padella.
Use DIG-109 toxin and DIG-152 toxin and variant thereof with the coleopteran pest of control crop plants also among expecting.In some embodiments; Cry albumen can be disposed at the control of insect pest economically, includes but not limited to, for example; Rootworm is Diabrotica undecimpunctata howardi (southern corn rootworm) for example; Diabrotica longicornis barberi (northern corn rootworm) and Diabrotica virgifera (west corn rootworm), and maggot Cyclocephala borealis (northern masked chafer) for example; The larva of Cyclocephala immaculate (southern masked chafer) and Popillia japonica (Japanese beetle).
The antibody test of DIG-109 and DIG-152 toxin
t antibody.The disclosed antibody of this paper to the B.t. toxin, or to equivalent toxin, or the fragment of these toxin, can come easily to prepare through the standard procedure of this area, for example by Coligan et al., 2007 instruction and renewal thereof.Such antibody is of great use to the existence that detects DIG-109 toxin, DIG-152 toxin and variant thereof.
In case the B.t. Pesticidal toxins is separated, the special antibody of contratoxin just can obtain through conventional method well known in the art.The time that continues several weeks or several months to host's duplicate injection of selecting will cause immune response and cause significant resisting-B.t. toxin serum titer.Preferred host is a mammalian species, and preferred species are rabbits, goat, sheep and mouse.The blood of from these immune animals, obtaining can through ready-made method handle with obtain can with the antiserum (polyclonal antibody) of B.t. Pesticidal toxins reaction.Antiserum can carry out the affinity purifying according to technology known in the art through being adsorbed to toxin then.The antiserum of affinity purifying can be further purified through the immunoglobulin part that uses methods known in the art to separate in the antiserum.The material that obtains is the allos population with the immunoglobulin of B.t. insecticidal proteins reaction.
Anti--B.t. toxin antibody can be puted together the semi-synthetic immunogene that forms by the synthetic property fragments of peptides of B.t. Pesticidal toxins and immunogenic carrier through preparation and produce.The kinds of schemes and the means of making fragments of peptides all are well known in the art.A lot of suitable immunogenic carriers for example bovine serum albumin(BSA) or Keyhole Limpet hemocyanin also are well known in the art, and the technology of coupling immunogene and carrier protein.In case semi-synthetic immunogene is fabricated, making the method for the antibody of B.t. Pesticidal toxins fragments specific is identical with the technology that is used to make the antibody that reacts with natural B .t. toxin.
Anti--B.t. toxin monoclone antibody (MAbs) can use the B.t. Pesticidal toxins of purifying and easily prepare.The method of generation MAb has been put into practice above 15 years and has been known to those skilled in the art.The B.t. Pesticidal toxins of purifying in adjuvant, repeats intraperitoneal or hypodermic injection will cause immune response in most of animals.From animal, shift out hyperimmune B-lymphocyte and merge with the suitable fusion partner cells system that can infinitely cultivate.Preferred animal is that the B-lymphocyte can be by hyperimmune and what be used to produce MAb is mammal.Preferred animal is rat and mouse and most preferably BALB/c mouse strain.
Multiple mammal cell line all is the suitable fusion partner that produces hybridoma.A lot of such cell-lines can (ATCC, Manassas VA) obtain with commercial provider from U.S. typical case's culture collecting center.Preferred fusion partner cells is to be the myeloma that is derived from mouse; And
Figure BDA00002019672300211
Friendly myeloma-653 cell-line (Ventrex; Portland is most preferred ME).In case after merging, the hybridoma of acquisition was cultivated for one to two week in the selective growth medium.Can use two known screening systems to come from bulk crossing knurl culture fluid, to get rid of the myeloma cell of not merging, or the fusion between the myeloma cell.The myeloma fusion partner of the strain and the use of mice immunized is depended in the selection of screening system.Can use Samloff, the aaT screening system that (1983) are described by Taggart and; Yet HAT (hypoxanthine aminopterin, thymidine) the screening system of being described by Littlefield (1964) is preferred, because more compatible with above-described preferred mouse strain and fusion partner.The growth medium of using screens immunologic opsonin MAb secretion subsequently.Enzyme Linked Immunoadsorbent Assay (ELISA) method is only to this purpose; However, the radioimmunoassay of suitable big volume screening also is an acceptable.Can be designed to reduce continuously the multiplex screening irrelevant or the lower culture that needs of considerable number.The culture of secretion and the MAb of B.t. Pesticidal toxins reaction can be through screening with the cross reaction of known B.t. Pesticidal toxins.The MAb that preferentially is bonded to preferred B.t. Pesticidal toxins can confirm type with commercial obtainable analytical method.Preferred L Ab is the IgG class, and preferred MAb is IgG 1And IgG 2aHypotype.
The hybridoma culture of secretion preferred L Ab can subclone repeatedly to set up monoclonicity and stability.The subclone eukaryotic, the known method that non-adherent cell is cultivated comprises restricted dilution, the cell sorting technology of soft agarose and fluorescent activation.Behind each subclone, the culture of acquisition is preferably analyzed antibody-secreting and type again to confirm to have set up stable preferred L Ab secretion culture.
Anti--B.t. toxin antibody can be used for detecting the several different methods of B.t. Pesticidal toxins, its variant and the fragment of requirement of the present invention.The antibody of a report of known usefulness component mark can be used for differentiating the existence of various environment antigens.The antibody of labelled with radioisotope has been used for the radioimmunoassay many decades, and it is used for analyzing the existence of various biotic habitat antigens and has very high accuracy and sensitivity.Recently, the antibody of enzyme labeling has been used as the alternative ELISA of the being used for detection of radio-labeled antibody.Further, can be bonded to immobilization matrix for example polystyrene hole or particle and be used for immunoassay to the immunoreactive antibody of B.t. Pesticidal toxins of the present invention to confirm whether the B.t. toxin is present in a specimen.
Detect used probe
The further method of differentiating toxin of the present invention and gene is to use oligonucleotide probe.These probes are detectable nucleotide sequences.These sequences can maybe can realize detectivity through intrinsic fluorescence through relying on suitable radioactive label, are described in the US patent No. 6268132.Known in this field, if probe molecule and nucleic acid samples are hybridized through forming two intermolecular strong base pairing keys, then can infer this probe and sample and have basic sequence homology.Preferably, hybridization is under stringent condition, to carry out through technology well known in the art, describes and for example Keller and Manak (1993).The detection of probe provides confirms the method whether hybridization takes place in a known manner.Such probe analysis provides the fast method of differentiating toxin encoding gene of the present invention.Nucleotide fragments as probe according to the present invention can use DNA synthesizer and standard procedure to synthesize.These nucleotide sequences also can be used as the PCR primer with the gene of the present invention that increases.
Hybridization
Biology field is well-known, and the similitude of two nucleic acid can characterize through the tendentiousness of their hybridization.Term as used herein " stringent condition " or " tight hybridization conditions " are meant such condition, and probe will be hybridized (annealing) extremely than the detectable higher degree of other sequence (for example, surpassing 2 times of backgrounds at least) with its target sequence with this understanding.Stringent condition be sequence dependent and under varying environment, be different.Stringency through control hybridization and/or washing condition can identify the target sequence (homology detection) with probe 100% complementation.Alternatively, stringent condition can change with some mispairing in the permission sequence, thereby can detect the more similitude of low degree (allos detection).Usually, probe is the length that is less than 1000 nucleotide, preferably is less than the length of 500 nucleotide.
Typically; Stringent condition is meant that wherein salinity is lower than 1.5M Na ion, typically about 0.01-1.0M Na ion concentration (or other salt) pH 7.0 to pH 8.3 and temperature be to short probe (for example 10-50 nucleotide) at least about 30 ℃ and to long probe (for example greater than 50 nucleotide) at least about 60 ℃.Stringent condition also can subtract for example formamide of stable reagent through interpolation.Exemplary low stringent condition comprises with following condition hybridization: buffer solution is the 30%-35% formamide; 1M NaCl; 1%SDS (lauryl sodium sulfate) under 37 ℃, and with 1X to 2X SSC (20X SSC=3.0M NaCl/0.3M trisodium citrate) 50 ℃-55 ℃ down washings.Exemplary medium stringent condition comprises with the hybridization of following condition: buffer solution is the 40%-45% formamide, 1.0M NaCl, 1%SDS (lauryl sodium sulfate) under 37 ℃, and with 0.5X to 1X SSC 55 ℃-60 ℃ washings down.Exemplary high stringent condition comprises with the hybridization of following condition: buffer solution is 50% formamide, and 1M NaCl, 1%SDS (lauryl sodium sulfate) and wash down at 60 ℃-65 ℃ with 0.1X SSC under 37 ℃.Alternatively, lavation buffer solution can comprise about 0.1% to about 1% SDS.Hybridization time is generally less than about 24 hours, about usually 4-12 hour.
Specificity typically is the function of post-hybridization washing, ion strength and temperature that its most important factor is final wash solution.To DNA/DNA hybridization, its thermal melting point (T m) be that 50% complementary target sequence is hybridized the temperature (under ion strength of confirming and pH) in the probe of Perfect Matchings.Every mispairing 1%, T mTo reduce about 1 ℃; Therefore, T m, hybridization conditions, and/or wash conditions can be adjusted into the annealing of the sequence that helps specifying homogeneity.For example, if search>The sequence of 90% homogeneity, T mCan reduce by 10 ℃.Usually, stringent condition is elected bit sequencing row and its complement as at ion strength of confirming and the T under the pH mLow about 5 ℃.Yet high stringent condition can use and be lower than T m1 ℃, 2 ℃, 3 ℃, or 4 ℃ hybridization and/or washing; Medium stringent condition can use and be lower than T m6 ℃, 7 ℃, 8 ℃, the hybridization and/or the washing of 9 ℃ or 10 ℃; And low stringent condition can use and is lower than T m11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃, or 16 ℃ hybridization and/or washing.
T m(in ℃) can be confirmed or calculate approx through experiment.For DNA-DNA hybridization, T mCan come to calculate approx from Meinkoth and Wahl equation (1984):
T m(℃)=(%GC)-0.61,81.5 ℃+16.6 (log M)+0.41 (% formamide)-500/L;
Wherein M is the molar concentration of monovalention, and %GC is the percentage of guanine and cytidylic acid among the DNA, and the % formamide is the percentage of formamide in the hybridization solution, and L is the length of hybridization in base-pair.
Alternatively, T mBy following formula (Beltz et al., 1983) are described.
T m(℃)=81.5 ℃+16.6 (log [Na+])+0.41 (%GC)-0.61 (% formamide)-600/L
Wherein [Na+] is the molar concentration of sodium ion, and %GC is the percentage of guanine and cytidylic acid among the DNA, and the % formamide is the percentage of formamide in the hybridization solution, and L is the length of hybridization in base-pair.
Use these equations, hybridization and cleaning compsns, and the T that needs m, those skilled in the art will appreciate that the variation of the stringency of hybridization and/or wash solution is described by constitutionally.Mispairing degree if desired causes T mBe lower than 45 degrees centigrade (aqueous solution) or 32 ℃ (formamide solution), preferably increase SSC concentration can use higher temperature.To the instruction widely of the hybridization of nucleic acid referring to Tijssen (1993) and Ausubel et al., 1995 and upgrade.Also referring to Sambrook et al., (1989) and upgrade.
Immobilized DNA can pass through standard technique with the hybridization of radiolabeled gene-specific probe on southern blotting technique (Sambrook et al. supra.) carries out.The radioisotope that is used for the mark polynucleotide probes can comprise 32P, 33P, 14C, or 3H.Radioisotope be integrated into the polynucleotide probes molecule can by the accomplishing arbitrarily of the known several different methods of biology field technical staff (referring to, for example, Sambrook et al., supra.).Usually, hybridization and subsequent washing can be carried out under such stringent condition, the target sequence that the encoding gene of the toxin that it allows to detect and require has autoploidy.To the double-stranded DNA gene probe, hybridization can be at the T that is lower than DNA hybridization mSpend the night T wherein under 20-25 ℃ mBe at 6X SSPE, 5X Denhardt's solution, 0.1%SDS, the T of DNA hybridization under the 0.1mg/mL denatured DNA m[20X SSPE is 3M NaCl, 0.2M NaHPO 4, and 0.02MEDTA (disodium edta); 100X Denhardt's solution is the 20gm/L polyvinylpyrrolidone, 20gm/L ficoll 400 (Ficoll type 400) and 20gm/L bovine serum albumin(BSA) (fragment V)].
Washing is typically carried out as follows:
Carried out (low tight washing) among the 0.1%SDS at 1X SSPE in 2 times at room temperature 15 minutes.
1 time at T m-20 ℃ following 15 minutes at 0.2X SSPE, carry out (high tight washing) among the 0.1%SDS.
To oligonucleotide probe, hybridization can be lower than the T of hybridization mSpend the night T wherein under 10-20 ℃ mBe at 6X SSPE, 5X Denhardt's solution, 0.1%SDS, the 0.1mg/mL denatured DNA is the T of hybridization down mThe T of oligonucleotide probe mCan confirm (Suggs et al., 1981) through following formula.
T m(℃)=2 (number of T/A base-pair)+4 (number of G/C base-pair)
Washing is typically carried out as follows:
Carried out (low tight washing) among the 0.1%SDS at 1X SSPE in 2 times at room temperature 15 minutes.
1 time under hybridization temperature 15 minutes at 1X SSPE, carry out (higher tight washing) among the 0.1%SDS
Some instances of the combination of salinity and temperature are (in order to increase stringency) as follows: 2X SSPE or SSC are at room temperature; 1X SSPE or SSC are under 42 ℃; 0.1X SSPE or SSC are under 42 ℃; 0.1XSSPE or SSC is under 65 ℃.
The hybrid molecule that forms between the probe molecule of hybridization and probe and the target molecule can present the detectability of the mode outside the radioactive label.Such optional method is also attempted within scope of the present invention.
Be to be understood that embodiment described herein and embodiment are only for exemplary purposes, and its various modifications or change and will advise and be included within the application's spirit and the scope and within the scope of appended claim by those skilled in the art.
Only if specialize or hint, the term " (a) " that this paper uses, " one (an) " and " these (the) " means " at least one ".
Be the embodiment of explanation practice mode of the present invention below.These instances should not be interpreted as restricted.All percentages are by weight, and all solution mixture ratios are by volumes, unless stated otherwise.All temperature are Celsius temperatures.
Embodiment 1
Design chimeric Cry1Ca active toxin and Cry1Ab parent toxin
chimeric toxin.The chimeric protein that merges with the parent toxin fragment of the active toxin domain of a Cry toxin and another Cry toxin before be in the news for example the US patent No. 5593881 and the US patent No. 5932209.A Cry1Ca3 δ exotoxin protein sequence leaves GenBank registration number AAA22343 in, under an old title of being CryIC (b).
Cyr1Ca chimeric protein variant of the present invention comprises such toxin, and it comprises N-end core toxin fragment and allos δ exotoxin parent toxin fragment position behind several points after the end of said active toxin fragment of being derived from the Cry1Ca3 Pesticidal toxins and merges.Connect perhaps to active toxin/parent toxin that the transformation of allos parent toxin fragment can occur in approximately self from active toxin; Alternatively; The part (extend through active toxin fragment) of self parent toxin can be retained, and occurs in downstream to the transformation of allos parent toxin.In variant type, active toxin and parent toxin fragment can accurately comprise the amino acid sequence of self toxin in its source, can comprise that perhaps amino acid adds; Deletion, or replace, it can not reduce; Perhaps possibly strengthen the biological function of said fragment, in the time of itself and another fusion.
For example, chimeric protein of the present invention comprises active toxin fragment and the allos parent toxin that is derived from Cry1Ca3.In a preferred implementation of the present invention; Be derived from the active toxin fragment of Cry1Ca3; As disclosed among the SEQ ID NO:1 (619 amino acid), and comprise the allos fragment that is derived from Cry1Ab δ-ectotoxic parent toxin fragment and merge as Cry1Ca active toxin fragment.SEQ ID NO:2 discloses 545 amino acid sequences of a parent toxin fragment that is derived from Cry1Ab and has been used for Cry1Ca variant of the present invention.Note last about 100-150 amino acid of this parent toxin fragment of SEQ ID NO:2, with its be included in chimeric toxin of the present invention within be very important.Thereby a preferred implementation of the present invention comprises such chimeric protein, and wherein the disclosed Cry1Ca active toxin of SEQ ID NO:1 fragment is incorporated into the parent toxin fragment of the disclosed Cry1Ab of being derived from of SEQ ID NO:2.1164 amino acid sequences of this chimeric protein, this paper is called DIG-152, in SEQ ID NO:3 open (pMYC2547 version).Of the present invention second preferred embodiment comprises such chimeric protein, wherein the disclosed Cry1Ca active toxin of SEQ ID NO:1 fragment be incorporated into that SEQ ID NO:4 shows second the 545 amino acid whose parent toxin fragment that is derived from Cry1Ab.Note last about 100-150 amino acid of this parent toxin fragment, with its be included in chimeric toxin of the present invention within be very important.1164 amino acid sequences of this second chimeric protein, this paper is called DIG-109, open in SEQ ID NO:5 (version that corn is optimized).Be to be understood that comprise Cry1Ca active toxin variant be derived from Cry1Ab parent toxin other chimeric fusion also within the scope of the invention.
Notice that the parent toxin fragment that is derived from Cry1Ab that SEQ ID NO:2 and SEQ ID NO:4 show is a function equivalent each other basically, the difference on the sequence only is (a first) position.
Embodiment 2
Make up the expression plasmid of coding chimeric Cry1Ca core/Cry1Ab parent toxin albumen and at false unit cell Express in the bacterium
The standard clone technology [for example is described in Sambrook et al.; (1989) and Ausubel et al.; (1995); And upgrade] (Pseudomonas fluorescens, Pf) expression construct pMYC2547, this construct are processed as to produce and comprise the total length chimeric protein (DIG-152 that the Cry1Ca core is blended in the Cry1Ab parent toxin to be used to make up pseudomonas fluorescens; SEQ ID NO:3).In the pseudomonas fluorescens strain MB214 (strain of deriving of bacterial strain MB101; Pseudomonas fluorescens biovar I) carry out in, this bacterial strain has the lac operon of the modification of insertion, as disclosed in the US patent No. 5169760.Basic clone's strategy comprises that the dna fragmentation subclone with encoding D IG-152 advances plasmid vector, and wherein it places that (PL Pharmacia, Milwaukee is under Ptac promotor WI) and the control of rrnBT1T1 terminator from the pKK223-3 plasmid.Such plasmid is named as pMYC2547, and the MB214 with this plasmid of screening is named as Dpf108.
Shake growth and expression analysis in the bottleBe used to characterize with the production of the DIG-152 of insect bioanalysis and accomplish through a pseudomonas fluorescens strain Dpf108 who shakes the bottle growth.Operate as described in the production of the DIG-152 albumen of Ptac promoters driven such as the previous US patent No. 5527883.Shake 30 ℃ of initial cultivations of bottle and come abduction delivering through adding isopropyl-β-D-1-thiogalactoside (IPTG) after 24 hours.Culture is when inducing and induce the back different time to take a sample.Optical density (OD600) through at 600nm is measured cell density.
The cell separation and the SDS-PAGE that shake bottle sample analyzeIn each sample time, the cell density of sample is adjusted to OD 600=20, the part of 1mL under 14000xg centrifugal 5 minutes.The cell fritter is freezing down at-80 °.Use EasyLyse TMBacterioprotein extraction solution (
Figure BDA00002019672300271
Biotechnology, Madison WI) produces from the freezing solvable and insoluble part of shaking bottle cell fritter sample.Each cell fritter is resuspended in EasyLyse TMSolution also further dilutes with 1:4 in lysis buffer, and at room temperature vibration was hatched 30 minutes.Lysate under 14000rpm 4 ° centrifugal 20 minutes, reclaim supernatant as soluble fraction.Particle (insoluble part) is resuspended in isopyknic PBS (PBS then; 11.9mM Na 2HPO 4, 137mM NaCl, 2.7mM KCl, pH7.4).
Sample and the 2X Laemmli sample buffer that comprises beta-mercaptoethanol (Sambrook et al., supra.) mix with 1:1 and boil be splined on after 5 minutes Criterion XT Bis-Tris 12% gel (Bio-Rad Inc., Hercules, CA).In the XT MOPS buffer solution of recommending, carry out electrophoresis.Gel dyes according to manufacturer's (Bio-Rad) step with Bio-Safe Coomassie Stain and (San Leandro CA) takes a picture with Alpha Innotech imaging system.
The preparation of inclusion body.Carry out on the cell of the pseudomonas fluorescens fermentation that is prepared in production solubility B.t. insecticidal proteins of DIG-152 albumen inclusion body (IB), and verify through SDS-PAGE and MALDI-MS (substance assistant laser desorpted/MALDI-MS).Pseudomonas fluorescens fermentation thalline thaws in 37 ° of water-baths.Cell with 25%w/v be resuspended in lysis buffer [50mM Tris, pH 7.5,200mM NaCl, 20mM EDTA disodium salt (ethylenediamine tetra-acetic acid), 1%Triton X-100 and 5mM dithiothreitol (DTT) (DTT); The bacterialprotease inhibitor mixed thing of 5mL/L (numbering #P8465; Sigma-Aldrich, St.Louis MO) adds before use].Cell with the hand-held homogenizer the minimum set low suspension (Tissue Tearor, BioSpec Products, Inc., Bartlesville, OK).Lysozyme (the Sigma L7651 of 25mg, white from egg) adds cell suspending liquid through the metal spoon, and suspension was at room temperature hatched 1 hour.Suspension is used Branson Sonifier 250 (twice 1-minute process, in 50% load cycle, 30% output down) ultrasonication then cooled on ice 15 minutes.The cell lysate is used microexamination.Add extra 25mg lysozyme if necessary, repeat to hatch and the ultrasonication process.Confirm after the cell bacteriolyze through microscope, lysate under 11500xg centrifugal 14 minutes (4 °) to form IB fritter, abandoning supernatant.The IB fritter is resuspended with the 100mL lysis buffer, with hand-held blender homogenising and as previously mentioned centrifugal.The IB fritter repeats with resuspended liquid washing (in the 50mL lysis buffer), and homogenising is ultrasonic, centrifugally then becomes colourless and the IB fritter color stable and that turn white that becomes up to supernatant.After the last washing, the IB fritter is resuspended in the distilled water of the aseptic filtration (0.22 μ m) that comprises 2mM EDTA, and centrifugal.Last fritter is resuspended in the distilled water of the aseptic filtration that comprises 2mM EDTA, and stores down at-80 ° with the component of 1mL.
The SDS-PAGE of protein analyzes to dilute with 1:20 with the quantitative IB fritter through the 1mL component that thaws and with the distilled water of aseptic filtration and carries out in the IB preparation.The sample of dilution reduces sample buffer [250mM Tris with 4X subsequently; PH6.8; 40% glycerine (v/v); 0.4% bromophenol blue (w/v); 8%SDS (w/v) and 8% beta-mercaptoethanol (v/v)] boil and be splined on
Figure BDA00002019672300281
4-20%Tris-glycine, 12+2 hole gel (Invitrogen) runs glue with 1X Tris/ glycine/SDS (BioRad).Run glue down at 200 volts and used Coomassie blue (50%G-250/50%R-250 is at 45% methyl alcohol, in 10% acetate) dyeing in 60 minutes then, use 7% acetate then, the distilled water solution decolouring of 5% methyl alcohol.The quantitatively density value through contrasting this band and bovine serum albumin(BSA) (BSA) the standard band that on same gel, runs of target stripe is to produce calibration curve.
The dissolving of inclusion body.6mL is centrifugal so that inclusion body becomes fritter down in the little centrifugal setting (about 14000xg) the most at a high speed of Eppendorf model 5415C from the DIG-152 inclusion body suspension of Pf clone DPf108.Remove store buffer liquid supernatant and with the 100mM sodium carbonate buffer replacement of the pH11 of 25mL, in the 50mL conical pipe.Inclusion body with pipette resuspended and vortex with abundant mixing.Test tube places and under 4 °, spends the night on the platform that softly rocks with the extraction target protein.Extract under 30000xg 4 ° centrifugal 30 minutes, (30,000 molecular weight cut off the supernatant of acquisition with Amicon Ultra-15 regenerated cellulose spin-on filter device; Micropore) concentrates 5 times.Sample buffer change into then 10mM CAPS [3-(cyclohexylamine) 1-propane sulfonic acid] the replaceable PD-10 post of pH 10 usefulness (GE Healthcare, Piscataway, NJ).
The dissolving and the trypsase activation of inclusion body protein.In some instance, centrifugal so that inclusion body becomes fritter down in the little centrifugal setting (about 14000xg) the most at a high speed of Eppendorf model 5415C from the DIG-152 inclusion body suspensions of Pf clone DPf108.Removing store buffer liquid supernatant and using the 100mM CAPS replacement of pH11 is about 50mg/mL so that protein concentration to be provided.Test tube at room temperature shakes 3 hours with abundant solubilising protein.Trypsase adds with the amount that equals 5%-10% (w:w is based on the initial weight of IB powder) and under 4 °, spends the night or at room temperature shake and accomplished digestion in 90-120 minute through hatching to shake simultaneously.Through removing insoluble matter in centrifugal 15 minutes under the 10000xg, supernatant is used for MonoQ ion exchange column (10mm takes advantage of 10cm).The DIG-152 albumen of activation comes wash-out with 0% to 100% 1M NaCl gradient through 25 column volumes.Merge the part comprise activated protein, and need, be concentrated into and be less than 10mL with the foregoing Amicon Ultra-15 cellulose spin-on filter device of living again.Material is then through Superdex 200 posts (16mm takes advantage of 60cm), and used buffer solution comprises 100mM NaCl.10% glycerine, 0.5%Tween-20 and 1mM EDTA.Confirmed that through the SDS-PAGE analysis (the enzyme brachymemma) albumen eluent of activation is 65-70mL.Merging the part that comprises activated protein also concentrates with the described centrifugal inspissator of preceding text.
gel electrophoresis.The protein Preparation thing that concentrates is through using
Figure BDA00002019672300291
LDS sample buffer (Invitrogen) is used for electrophoresis with after the 1:50 dilution, said buffer solution contain 5mM DTT as go back original reagent and be heated to 95 ° 4 minutes.Sample is splined on the double swimming lane of 4-12%
Figure BDA00002019672300292
gel; The next door is 5 BSA standards, from the scope (being used for the production standard curve) of 0.2 μ g to 2 μ g/ swimming lane.The voltage of 200V is provided and runs glue buffer solution (Invitrogen) and arrive the gel bottom up to tracer dye with MOPS SDS.Gel is with 45% methyl alcohol of 0.2% Coomassie blue G-250 and the solution-dyed of 10% acetate, and decolouring, uses 45% methyl alcohol simply for the first time, and 10% acetate is used 7% acetate then for a long time, and 5% methyl alcohol is eliminated up to background.After the decolouring, gel scans with BioRad Fluor-S MultiImager.The Quantity One Software of this instrument v.4.5.2 be used to obtain to dye protein band the volume that goes background and produce the BSA calibration curve, it is used for calculating the concentration of the chimeric DIG-152 albumen of solution of storage.
Embodiment 3
The insecticidal activity of the DIG-152 that produces in the pseudomonas fluorescens
The insecticidal activity of DIG-152 albumen is verified on the Lepidoptera species, comprises European corn borer (ECB; Ostrinia nubilalis (H ü bner)), cry1F-resistance ECB (rECB), cotton bollworm (CEW; Helicoverpa zea (Boddie)), black cutworm (BCW; Agrotis ipsilon (Hufnagel)), autumn mythimna separata (FAW, Spodoptera frugiperda (J.E.Smith)), Cry1F-resistance FAW (rFAW) and southwestern corn borer (SWCB, Diatraea grandiosella).
sample preparation and bioanalysis.Inclusion body prepared product (albumen of self full-length proteins or trypsase activation) is for example dialysed through switching method or the PD-10 post changes in the 10mM CAPS pH10 buffer solution.Sample suitably is diluted among the 10mM CAPS pH10 then, and all bioanalysis comprise the control treatment that this buffer solution is formed, and it is as lethality or the contrast of growth inhibiting background.
The protein concentration of bioanalysis buffer solution is made with BSA through gel electrophoresis the calibration curve of gel density is assessed, and measures with the described BioRad imaging system of preceding text.Protein in the gel-in-matrix is used based on the dyeing of Coomassie blue and is being read preceding decolouring.
Purified proteins is tested insecticidal activity in bio-measurement, carry out on artificial insect food through nascent lepidopterous larvae.ECB, CEW, BCW, (hatch for Benzon Research Inc., Carlisle by the ovum that the clone who PA) keeps obtains by commercial insectarium for the larva of FAW and SWCB.The clone that the larva of rECB and rFAW is had by the individual (IN) hatch for Dow AgroSciences, Indianapolis by the ovum of results.
(C-D International, Pitman carry out on NJ) being specifically designed to 128 hole vinyl discs of insect bioanalysis in bioanalysis.Each hole contain 1.0mL many species Lepidoptera food (Southland Products, Lake Village, AR).The protein sample of 40 μ L components is delivered to the 1.5cm in every hole with pipettor 2Foodstuff surface (that is 26.7 μ L/cm, 2).Food concentration is calculated as the amount (ng) of the DIG-152 albumen of every square centimeter of surface area in the hole.The dish of handling remains in the fume hood up to the liquid evaporation of foodstuff surface or is absorbed entering food.
After sprouting wings several hours, pick up single larva and be deposited on the food of handling the larva in every hole with moistening camel hair brush.The hole of infecting seals with the adhesive membrane of clean plastics, and opens the air port to allow gas exchange (C-D International).The bioanalysis dish remains on that [28 °, about 40% relative moisture (RH), 16 hours: 8 hours (light: dark)] kept 5 days under the environmental condition of control, and all insects are exposed to every kind of protein sample, the weight of record dead insects quantity and survival insect after the time.Calculate the percentage lethality and the percentage growth inhibition of each processing.Percentage growth inhibition (GI) is used computes:
%GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)]x100
Wherein TWIT be insect gross weight in this processing ( TOtal WEight of INsects in the TReatment);
TNIT be insect sum in this processing ( TOtal NUmber of INsects in the TReatment);
TWIBC be background detect (buffer solution contrast) middle insect gross weight ( TOtal WEight of INsects in the BAckground CHeck), and TNIBC to be background detect in (buffer solution contrast) insect total ( TOtal NUmber of INsects in the BAckground CHeck).
GI 50Confirm as the %GI value and be the concentration of chimeric DIG-152 albumen in 50 o'clock food.LC 50The concentration of DIG-152 albumen in food when (50% dead concentration) is recorded as 50% test insect and is killed.(SAS, Cary NC) carry out statistical analysis (One-way ANOVA) with JMP software.
Table 3 has been showed the picked-up bioanalysis of DIG-152 albumen to the test insect larvae of type in 7.
Table 3.GI 50And LC 50Value (ng/cm 2), calculate from the insect food of top-loaded DIG-152 albumen.
Figure BDA00002019672300311
DIG-152 albumen of the present invention has this characteristic, and promptly the picked-up DIG-152 albumen that is grown in of the nascent larva of autumn mythimna separata (Spodoptera frugiperda) and southwestern corn borer (Diatraea grandiosella) is suppressed afterwards.Further, there is the autumn armyworm larvae of resistance the same with wild type autumn armyworm larvae responsive to Cry1F toxicity to DIG-152.
Embodiment 4
The further insecticidal activity of the DIG-152 albumen of producing by pseudomonas fluorescens
The Lepidoptera insecticidal activity of DIG-152 albumen (not being the trypsase activation) is further at sugarcane borer (SCB; Diatraea saccharalis) and on the nascent larva of Cry1Ab-resistance SCB (rSCB) confirm with the food integration method with the dose response experiment.The DIG-152 inclusion body softly rocks the 100mMCAPS pH11 that was dissolved in 7.5mL in 4 hours under 4 °, and 1mMEDTA has wherein added bacterioprotein enzyme inhibitor (the Sigma P4865 of 200 μ L; Explanation preparation according to supplier).Centrifugal making after the insoluble material formation fritter, the protein concentration of storage is adjusted to 4.0mg/mL at 100mM CAPS among the pH11.For the insect bioanalysis; The concentration of DIG-152 albumen in 0.030 μ g-102 μ g/gm food scope is through the food (Bio-Serv of suitable volume with the part chemistry; Frenchtown; NJ) mix and prepare, be dispensed to immediately afterwards in the food of individual cells of 128 porose discs (Bio-Ba-128, C-D International).
The Cry1Ab albumen of trypsase activation (as the positive control of insecticidal activity) is at the scope build-in test (distilled water through freeze-dried mixed powder and appropriate amount before food preparation prepares) of 0.03125 μ g-32 μ g/gm food.
With the food (blank is for the Cry1Ab test) of distilled water preparation or only buffer solution (100mM CAPS pH11 is for the DIG-152 test) as control treatment.A nascent larva of sugarcane borer (the hatching back 24 hours) be released on the foodstuff surface in every hole.After the larva inoculation, cell covers with cowling (C-DInternational), and the bioanalysis dish places environmental cabinet to keep 28 °, 50%RH and 16 hours: 8 hours (light: secretly) periodicity of illumination.At postvaccinal the 7th day record larval mortality, larva weight and do not prove the quantity of the survival larva of weight increase (every larva < 0.1mg).Each combination repetition of insect strain/>Cry protein concentration 4 times repeats with 16-32 larva at every turn.
The larval mortality standard is measured as " reality " lethality, and it considers dead (morbid state) larva and (short and small, as not take food) larva of survival, and it does not show significant body weight gain (be every larva < 0.1mg).The actual of larva calculates with this formula in a processing:
Actual (%)=[TDS/TNIT] x100
Wherein TDS be the dead larva number sum that adds short and small larva number ( TOtal number of DEad larvae plus the number of STunted larvae),
And TNIT be in this processing insect sum ( TOtal NUmber of INsects in the TReatment).
" reality " lethality (hereinafter referred is a lethality) to every kind of sugarcane borer strain is proofreaied and correct, and observed larval mortality is proofreaied and correct on the analysis Cry1Ab process result water blank food, and DIG-152 handles and then uses the only food of buffer solution processing.
Further analyze the dose response result of experiment to set up GI 50Value, [that is, larval growth suppresses the concentration that (%GI) value is a B.t. albumen in 50 o'clock food].The %GI value that comprises larva on the food of Cry1Ab albumen is used computes:
%GI=[TWC-TWT]/TWCx100
Wherein TWC is the TBW (Total body Weight of larvae feeding on water Control diet) of the larva of water control Food nursing, and
TWT is the TBW (Total body Weight of larvae feeding on Cry1Ab Treated diet) of the larva fed of food that Cry1Ab handles
Wherein, in order to calculate the result's who takes in as DIG-152 albumen larva %GI, use computes:
%GI=[TWB-TWT]/TWBx100
Wherein TWB is the TBW (Total body Weight of larvae feeding on Buffer-Only control treated diet) of the larva fed of the food of only buffer solution control treatment, and
TWT is the TBW (Total body Weight of larvae feeding on DIG-152 Treated diet) of the larva fed of food that DIG-152 handles
Do not increase (every larva < 0.1) if there is larva to have significant weight, then be appointed as 100% larval growth and suppress.With two-way ANOVA with insect strain and Cry concentration as two main factorial analysis growth inhibition data.LSMEANS check is used to confirm the difference under the level of α=0.05, handled.
Food on the sugarcane borer larva combines the result of bioanalysis to be shown in table 4.
The larval mortality and the growth inhibition (% mean+/-standard error) of the Cry1Ab-responsive (SCB) that table 4. is fed with the food that contains Cry1Ab or DIG-152 albumen and the corn borer dose response of Cry1Ab-resistance (rSCB) a
Figure BDA00002019672300331
Figure BDA00002019672300341
aRunning through all in one row, to handle the mean value of all following a same letter in the back be not (the P of significant difference<0.05; The LSMEANS check).The standard error of sem=mean value.
bμ g albumen/gm food
cDefine in the measurement of larval mortality as indicated.
dThese percentages are calculated with the formula described in the literary composition.
eThese percentages are calculated with the formula described in the literary composition.
fNT=does not detect
Data analysisCorrected dosage/lethality data are used for the probable value analysis then to confirm to cause 50% lethality (LC 50) value and the processing protein concentration of corresponding 95% confidence interval (CI).Used processing comprises the maximum concentration that produces the zero death rate in the probable value analysis, causes the least concentration of 100% lethality, and all results between these extreme values.The resistance ratio is through the LC of rSCB strain 50Value is calculated divided by the value of SCB insect.The lethal dose ratio testing is used for confirming whether the resistance ratio is remarkable on α=0.05 level.Analyze the lethality data with two-way ANOVA, under the level of α=0.05, confirm the difference of processing then with the LSMEANS check.Analysis result is showed in table 5.
Table 5. has combined the summary of the bioanalysis check of SCB and rSCB larva in the insect food of DIG-152 albumen or Cry1Ab albumen.
Figure BDA00002019672300342
aDefinition described in the measurement as indicated of larval mortality.
bThe resistance ratio of letter " S " is significant, and letter " NS " is inapparent, on 5% level and based on lethal dose, checks.
DIG-152 albumen of the present invention has following characteristic; Promptly after DIG-152 albumen is absorbed with similar level with the Cry1Ab albumen with activation; The growth of nascent sugarcane borer (Diatraea saccharalis) larva is suppressed; Perhaps larva is killed, and wherein the Cry1Ab albumen of activation causes same biologically.The further characteristic of DIG-152 is that the toxic effect to Cry1Ab albumen has the sugarcane borer larva of resistance still responsive to the toxicity of DIG-152 albumen.
Embodiment 5
To the rabbit polyclonal of chimeric Cry1Ca protein immunization reaction and the generation of mouse polyclonal antibody
Developed the variant of antibody with detection and quantification Cry1Ca chimeric protein and Cry1Ca chimeric protein, for example, in extract by the genetically modified plants preparation of producing albumen of the present invention.Standard immunoassay trace preparation/analytical method and ELISA method be used to characterize antibody and B.t. Protein Detection (for example, at Coligan et al., in 2007 instruction and upgrade).
The generation of polyclonal antibody.The proteantigen that is used for polyclone immunity is the DIG-152 albumen produced in the pseudomonas fluorescens described of embodiment 2 active toxin through the trypsase brachymemma.In addition, two peptides of Cry1Ca active toxin fragments specific are conjugated in that hemocyanin (Keyhole Limpet Hemocyanin) is gone up and as immunogene.Said peptide is corresponding to the 436-445 amino acid (VQRSGTPFLT of SEQ ID NO:1; Cry1Ca436; SEQ ID NO:6) and 591-600 amino acid (SEQPLFGAGS; Cry1Ca591; SEQ ID NO:7).To be accredited as Cry1Ca be unique to these peptide sequences when the protein sequence of Cry1Ca and the contrast of other Cry1 B.t. albumen of several types.Further, expect that these peptides are exposed to the surface of self Cry1Ca albumen.
Normal process through the seller that concludes a bargin carries out immunity and serum collection.(Princeton NJ) obtains polyclonal antibody through Covance.NZw is used to produce the polyclonal antibody to the DIG-152 albumen of trypsase activation.Immunity and serum have 14 days circulation timei between collecting.Dosage begins with albumen that comprises 0.5mg or the Freund's complete adjuvant of puting together peptide.Injection incomplete Freund's adjuvant preparation subsequently.
Merging from the serum of this two rabbits producing a collection of albumin A antibody purified (being called DIG152RPC1), its can with the albumino reaction of Cry1Ca active toxin.Known like antibody representational field technical staff, the polyclonal antibody that is produced by intact proteins is not very special usually and detects through regular meeting and to be used for the albumen of immunity and a lot of epi-positions of other GAP-associated protein GAP.Therefore, immunoassay shows that DIG152RPC1 can detect the B.t. toxin of other Cry1-class, especially, and the Cry1Ab of trypsase activation, Cry1Da, and the Cry1Be and the Cry1Ea of Cry1Fa and chymotrypsin protein enzyme activation.Notice that in commerce is provided with crop plants can produce other Cry1-plastein, so DIG152RPC1 demonstrates the useful reagent that is used to detect these albumen, comprise the brachymemma and other form of these albumen.
Developed the rabbit polyclonal antibody that to Cry1Ca two put together peptide specific batch.Two NZws are respectively applied for every kind of peptide, to every kind of peptide combining anteserum; Obtain two kinds of peptides a peptide antibody separately.Immunity is collected with serum and is carried out through normal process, 14 days circulation timei of interval between immunity and serum are collected.Final batch of peptide affinity purifying of serum with correspondence.The direct ELISA assessment of two kinds of peptide specific antibody shows that the antibody to peptide Cry1Ca591 demonstrates specific detection Cry1Ca, than with the reaction of other Cry1 plastein, and be not specific (table 6) to the antibody of peptide Cry1Ca436.
The direct ELISA optical density readings that two kinds of Cyr1Ca peptide specifics of table 6. antibody obtains with 1g/mL and various Cry1 B.t. proteantigen reaction back.
Cry1Ca Cry1Ad Cry1Fa Cry1Be Cry1Da Cry2Aa Cry1Ab Cry1Ea
Anti--Cry1Ca591 1.36 0.32 0.27 0.27 0.3 0.2 0.51 0.23
Anti--Cry1Ca436 0.39 0.32 0.41 0.42 0.54 0.32 0.81 0.38
MONOCLONAL ANTIBODIES SPECIFIC FOR.(Huntsville AL) prepares monoclone antibody through Open BioSystems/Thermo Fisher Scientific.The active toxin through the trypsase brachymemma of the DIG-152 protein Preparation of producing in the pseudomonas fluorescens of describing among the embodiment 2 is adopted in the exploitation of mouse anti-Cry1Ca monoclone antibody.The development of immunity and cell-line is carried out in cell culture medium through the standard antibody development approach, rather than through the ascites production method.Monoclonal cell system merges immune mouse boosting cell and the compatibility ND4 mouse myeloma cell line of crossing through normal process and obtains.
Directly combine the ELISA screening to identify mouse M4 serum and have remarkable specificity (table 7) Cry1Ca albumen.
The direct terminal point titre that combines ELISA to react of table 7. mouse M4 serum (with the Cry1Ca of trypsase activation immunity) and the albumen of several kinds of Cry1 classes.
Antigen Cry1Ca Cry1Da Cry1Ac Cry1F Cry1Be Cry1Ab
The end point titre 312500 62500 500 12500 <100 500
All monoclonal cells systems that are derived from M4 are through being bonded to Cry1Ca, Cry1Da, and Cry1Ac, Cry1Fa, the direct combination ELISA of Cry1Be and Cry1Ab tests.Cell-line M4-34 and M4-23, it is proved to be has the ability [that is, having high optical density (OD) reading] that detects Cry1Ca, and does not detect other Cry1 plastein [that is, having zero or low-down OD reading], is special (table 8) be concerned about.From preferred cell is that the monoclone antibody of M4-34 is called as antibody DIG152MabM4-34.
The optical density readings of the monoclonal cell system that table 8. is derived from M4 and the direct ELISA of combination of the Cry1 plastein reaction of the Cry1Ca albumen of the trypsase activation of target and non-target.
Antigen Cry1Ca Cry1Be Cry1Ac Cry1F Cry1Ab Cry1Da
Cell-line M4-34 2.024 0.029 0.105 -0.013 -0.008 0.03
Cell-line M4-23 1.799 0.07 0.095 0.061 0.043 0.064
Therefore an object of the present invention is to provide the monoclone antibody of the Cry1Ca B.t. albumen of specific recognition brachymemma.
Embodiment 6
The codon optimized sequence of corn of design encoding D IG-109 albumen
The molecular biology of plants those skilled in the art can understand a plurality of dna sequence dnas and can be designed as a kind of amino acid sequence of coding.The common implication of the expression of the coding region of increase protein of interest is that coding region is changed into such mode, and promptly its codon is formed similar host's overall codon composition, and gene is bound to express therein.About design and the instruction that produces synthetic property gene referring to the for example WO1997/13402 and the US patent No. 5380831.
Designed and synthesized dna sequence dna in the transgenosis monocotyledon, to produce the chimeric insecticidal proteins of DIG-109 with corn preference codon.The codon that 706 albumen coded sequences that obtain according to the sequence of depositing from GenBank (www.ncbi.nlm.nih.gov) have calculated corn (Zea mays L.) uses table.Ignoring any use is less than and is used for calculating the weighted average that the corn codon is provided with after this amino acid whose codon unnecessary codon of about 10% altogether.Represent the weighted average of each codon to use computes:
Weighted average %=1/ (%C1+%C2+%C3+etc.) x%C1x100 of C1
Wherein C1 is the codon of being discussed, and %C2, %C3, etc. represent the average % use value of all the other synonym.
The codon optimized dna sequence dna of corn for 1164 amino acid whose DIG-109 albumen of the SEQ ID NO:5 that obtains to encode; The codon that has carried out self cry1CaDNA sequence of coding Cry1Ca active toxin fragment replaces, and forms with the whole codons that obtain to have the preference password sublist that corn optimizes.With similar form, the codon of self cry1Ab dna sequence dna that has carried out the Cry1Ab parent toxin fragment of coding SEQ ID NO:4 replaces, and forms with the whole codons that obtain to have the preference password sublist that corn optimizes.To the carrying out of sequence further sophistication to eliminate undesirable Restriction Enzyme recognition site; Potential plant introne splice site; The A/T of long section or C/G residue and other maybe the disturb plant cell in rna stability, transcribe or translate the motif of coding region.Carry out other and changed the Restriction Enzyme recognition site that needs with introducings, and eliminated the inherent ORFs grown (be different from+1 reading frame).These change all and under the constraint that keeps corn preference codon combination roughly, carry out.The codon optimized sequence of complete corn of encoding D IG-109 albumen is open as SEQ ID NO:8.(DNA2.0, Menlo Park CA) carries out through commercial supplier according to the dna fragmentation of SEQ ID NO:8 synthetic.
Embodiment 7
Structure comprises the plant conversion carrier of the plant-expressible gene of encoding D IG-109 albumen
(Japan Tobacco, Tokyo JP) are used for monocotyledon host's conversion to the super double element system of Agrobacterium expediently.This super double element system uses the pSB11 shuttling expression plasmid vector, and it contains by isolated T-DNA right margin repetition of MCS (RB) and T-DNA left margin repetition (LB).A derivative (being called pDAB7691) that has prepared pSB11 through the standard DNA cloning process.Plasmid pDAB7691 comprises the DIG-109 coded sequence (CDS that corn is optimized; That is transcribing under the control and connect introne 1 (the US patent No. 5510474) and corn Per5 3 ' untranslated region territory (3'UTR) (the US patent No. 7179902) SEQ ID NO:8) in corn ubiquitin 1 promotor.Further; PDAB7691 contains the plant selectable marker gene, comprises Dow AgroSciences DSM2 CDS (WO 2008/070845 A2) transcribing under the control and connect introne 1 (the US patent No. 5641876) and corn lipase 3 ' UTR (the US patent No. 7179902) in rice actin 1 promotor.The concise and to the point signal as follows of physical arrangement of the component in the T-zone of pDAB7691:
RB>corn Ubi1 promotor: DIG-109 CDS: corn Per5 3 ' UTR>paddy rice Act1 promotor: DSM2 CDS: corn Lip 3'UTR>LB
Second derivative (being called pDAB100276) that has prepared pSB11 through the standard DNA cloning process.Plasmid pDAB100276 comprises the DIG-109 coded sequence (CDS that corn is optimized; That is transcribing under the control and connect introne 1 and corn Per5 3 ' UTR SEQ ID NO:8) in corn ubiquitin 1 promotor.Further, pDAB100276 contains the plant selectable marker gene, comprises Dow AgroSciences AAD1 CDS (US number of patent application 20090093366), transcribing under the control and connect introne 1 and corn lipase 3 ' UTR in corn ubiquitin 1 promotor.The concise and to the point signal as follows of physical arrangement of the component in the T-zone of pDAB100276:
RB>corn Ubi1 promotor: DIG-109 CDS: corn Per5 3'UTR>corn Ubi1 promotor: AAD-1 CDS: corn Lip 3'UTR>LB
In order to prepare the conversion of Agrobacterium, the escherichia coli cell clone strain DH5 α with plasmid pDAB7691 or plasmid pDAB100276 under 37 ° at the LB agar medium (g/L: bacto-tryptone, 10 that contains spectinomycin (100 μ g/mL); Bacterium is used yeast extract, and 5; NaCl, 10; Agar, 15) middle grow overnight.Contain and combine the bacterial strain DH5 α cell of transferring plasmid pRK2013 on the LB agar that contains kanamycin (50 μ g/mL), to grow.Flat board is hatched under 4 ° to wait the agrobacterium tumefaciens bacterial strain LBA4404 that contains plasmid pSB1 and can be used.
Embodiment 8
Transform Agrobacterium to produce super binary vector
The super double element system of Agrobacterium, its use contain the agrobacterium tumefaciens bacterial strain LBA4404 of plasmid pSB1, can be used for the transforming monocots host expediently.Make up and confirm that the methodology of super binary vector is set up well, referring to the operation manual (Japan Tobacco) of pSB1.Standard microorganism is learned and molecular biology method is used for producing and confirming super double base plasmid pDAS5162; This is the cointegrate that comprises plasmid pSB1 and pDAB7691; And super double base plasmid pDAS5848, this is the cointegrate that comprises plasmid pSB1 and pDAB100276.
Embodiment 9
In corn plant, produce DIG-109
The conversion of agriculture bacillus mediated cornFrom the seed of Hi-II F1 hybridization (Armstrong et al., 1991) be tiled in contain 95% Metro-Mix 360 soilless culture mediums (Sun Gro Horticulture, Bellevue, WA) with 5 gallon cans of the mixture of 5% clay loam in.Plant grows in the greenhouse, uses high-pressure sodium and metal chloride lamp with illumination in 16 hours: the photoperiodic combination of 8 hours dark.Close pollination under controlling is used for transforming to obtain jejune F2 embryo.Ripe embryo is not become in pollination back after about 10 days harvesting corn fringe when being big or small between the 1.0mm-2.0mm.
are infected and are cultivated altogether.Corncob shells and cleans surface sterilizing through liquid soap, is immersed in 20% commercial bleaching agent (containing 5% clorox) and keeps 20 minutes, uses rinsed with sterile water then 3 times.The suspension that comprises the agrobacterium tumefaciens cell of pDAS5162; Gene and the super double base plasmid that contains DSM2 plant selectable marker gene with encoding D IG-109 albumen; Bacterium through shifting 1 or 2 circulation [is containing the 100mg/L spectinomycin; The YEP solid culture medium of 10mg/L tetracycline and 150mg/L streptomycin (g/L: bacterium is used yeast extract, and 10; Bactopeptone, 10; NaCl, 5; Agar, 15) last 28 ° of growths 2-3 days down] infect medium [LS Basal medium (Linsmaier and Skoog, 1965) to the liquid that contains 100 μ M acetosyringones of 5mL; N6 vitamin (Chu et al., 1975), 1.5mg/L2; The 4-dichlorphenoxyacetic acid (2,4-D), 68.5g/L sucrose; 36.0g/L glucose, 6mM L-proline, pH 5.2] prepare.
Alternatively; The suspension that comprises the agrobacterium tumefaciens cell of pDAS5848; Gene and the super double base plasmid that contains AAD-1 plant selectable marker gene with encoding D IG-109 albumen are prepared through the bacterium that grows as stated to the liquid infection medium that contains 100-200 μ M acetosyringone of 5mL that shifts 1 or 2 circulation.
Under two kinds of situation; The vibration of solution vortex is up to the suspension that forms homogeneous; Using the Klett-Summerson colorimeter to regulate concentration to final concentration with the purple filter is 200 Klett units (pDAS5162 is transformed), or optical concentration 550nm place is that 1.2 (to pDAS5848) transform.The immature embryo direct separation is to the micro-centrifuge tube that contains 2mL infection medium.Remove medium and replace, Agrobacterium/embryo solution incubated at room 5-10 minute with the Agrobacterium solution of 1mL.Indusium changes the common medium of the acetosyringone (pDAS5848 is transformed) that contains 100 μ M acetosyringones (pDAS5162 is transformed) or contain 100-200 μ M over to [1.5mg/L 2 for LS Basal medium, N6 vitamin then; 4-D; 30.0g/L sucrose, 6mM L-proline, 0.85mg/L AgNO 3, the 2.8g/L gellan gum (KS), pH 5.8 for PhytoTechnology Laboratories, Lenexa], cultivated 3-4 days in 20 ° of following dark.
After cultivating altogether, embryo is transferred to and contains MS salt and vitamin, 6mM L-proline, and the 100mg/L inositol, 500mg/L MES, 30g/L sucrose, 1.5mg/L 2,4-D, 0.85mg/LAgNO 3, 250mg/L CTX (Cefotaxime), the 2.8g/L gellan gum is in the dormancy medium of pH 5.8.After about 7 days, embryo is transferred to and has replenished 3mg/L bialaphos (pDAS5162 is transformed) or replenished in the same medium of 100nM pyrrole fluorine chlorine standing grain spirit (haloxyfop) (pDAS5848 is transformed) (selection medium).Thereby the individuality that about 8 week backs transform is differentiated out and through being transferred to the swell regeneration and analyzing of fresh screening culture medium 2 time-of-weeks.
regeneration and seed produce.For regeneration, culture is transferred to " 28 " inducing culture (MS salt and vitamin, the 30g/L sucrose that has replenished 3mg/L bialaphos (pDAS5162 is transformed) or replenished the spirit of 100nM pyrrole fluorine chlorine standing grain (pDAS5848 is transformed); The 5mg/L benzylaminopurine; 0.25mg/L 2,4-D, 250mg/L CTX; 2.5g/L gellan gum, pH 5.7).Low light is according to (14 μ Em under the condition -2s -1) hatched for 1 week (about 89 μ Em under the high then illumination condition -2s -1) 1 week.Organize subsequently and be transferred to " 36 " medium (identical with inducing culture) of living again except lacking the plant growth regulating factor.When plantling is 3-5cm when long, it is transferred to contain SHGA medium [(Schenk and Hildebrandt (1972) salt and vitamin; PhytoTechnologies Labr.), the 1.0g/L inositol, 10g/L sucrose and 2.0g/L gellan gum, pH 5.8] the glass culture test tube in to allow the growth of further growth and bud and root.Plant is transplanted as in the said identical soil mixture of preamble and in the greenhouse, grow to and bloom.The pollination of controlling is to obtain seed.
The technical staff in corn conversion field will understand other method corn conversion and screening conversion plant also will be fine, when using the effable selected marker of other plant (for example herbicide tolerant gene).
Embodiment 10
Produce the biochemical analysis and the insect bioanalysis of the corn plant of DIG-109 albumen
The production of DIG-109 albumen in rotaring gene corn plant detects from the albumen of the leaf extraction of plant seedlings (T0 generation).The maize leaves disk of two 6mm diameters is placed in the sample tube of deep hole 96 cluster test tube casees (Costar Cat#3957), is chilled in-80 ° up to analyzing that day.At this time, two parts of Daisy that 4.5mm zinc encapsulates TMBB ' s adds each (freezing) test tube, and together with the extraction buffer solution of 200 μ L, said buffer solution comprises PBS (phosphate buffer; Fisher Cat#BP665-1) adds 0.05% polysorbas20.Cover each test tube, the test tube case places bead mill (Kleco TM4-96 Pulverizer; Garcia Manufacturing, Visalia CA) is provided with following 3 minutes in maximum.The sample of pulverizing under 2500xg centrifugal 5 minutes, the supernatant that contains soluble protein is used for immunoassay.
The immunoblotting assay of the maize leaves albumen that extracts show the DIG152RPC1 polyclonal antibody not with the albumen generation cross reaction of from the non-transgenic plant leaf, extracting.In the extract of pDAS5162 plant transformed, several kinds of albumen kinds have been arrived through the DIG152PRC1 antibody test.Have at least 4 main immune response bands to be detected routinely.Under a lot of situation, can see the albumen kind of a maximum, it moves with mobility corresponding to about 70kDa albumen.Other main albumen kind has the molecular size that is estimated as 65kDa (identical with the trypsase restriction peptide of the DIG-152 of Dpf108 preparation among the embodiment 2), 60kDa and 55kDa.When pDAS5162 transgenic corns leaf extract detected with the Western blotting of DIG-152 polyclonal antibody, the kind of 60kDa and 55kDa was a maximum in some plants.For any antibody, all have only the minority plant to have total length DIG-109 (130kDa) albumen, and even found, it also occur with very less important kind.
Obviously, although transform the transgenes encoding total length DIG-109 albumen of introducing corn through pDAS5162, the proteolytic activity in the maize cell is processed as sufficient stable small-molecular weight kind with nascent protein.
The insect toxicity of the leaf of from the rotaring gene corn plant with the conversion of pDAS5162 construct of independent separate, gathering in the crops carries out vitro detection through nascent larva of autumn mythimna separata (FAW, fall army worm (Spodoptera frugiperda) (J.E.Smith)) and the larva of Cry1F-resistance FAW (rFAW).The FAW ovum obtains from commercial supplier (Benzon), and the rFAW ovum is from patentee's population (Dow AgroSciences).Be used for the insect bioanalysis from the T0 plant of greenhouse growth migrating to the blade sections sample of greenhouse after about 2 weeks from the laboratory.Two blades of every strain plant (1 square inch of every agreement that contracts a film or TV play to an actor or actress) place the hole of the separation of 32 porose discs (CD International), on solidification 2% agar of about 3mL.Ovum hatches on multiple type of Lepidoptera food (Southland Products), selects the nascent larva less than 24 hours.About 10 larvas of every blade sections carefully are placed in every hole with camel hair brush, and the dish that infects remains on 28 ° with the lid may enclose with holes that dish provides, 40%RH, illumination in 16 hours: 8 hours dark 3 days.Write down the percentage injury (%DAM) of each blade during EOT.Average injury rate is used for confirming that which plant of test insect to every type has minimum injury.The test repetition of all insects for several times.
Data with the JMP statistical software (SAS, Cary NC) analyze, to every kind of insect type, the %DAM mark of average every plant species." Fit Y by X " model is used for individual event ANOVA and analyzes.Use the Tukey-Kramer average to separate when need analyzing the significant difference between the average %DAM mark of every kind of processing.Contrast with the %DAM mark that obtains from the check plant at similar age.The positive control plant is by commercial Herculex I TMThe heterozygote seed growth, it produces Cry1Fa B.t. toxin.Negative control (being non-conversion plant) is by Hi II and B104 system and a Herculex I TMVariation Lines (said Herculex I TMThe female parent that does not contain Cry of heterozygote) representative.
Fig. 1 has summed up the result that these insect bioanalysis tests obtain.This is an amazing discovery, i.e. the production of DIG-109 and the positive correlation between the %DAM ratio in the transgenosis leaf.For FAW, F=35.3; D.f.=1,33; P<0.0001; r 2=0.52, and for rFAW, F=25.3; D.f.=1,33; P<0.0001; r 2=0.43.Further surprising is that all autumn armyworm larvaes to Cry1FaB.t. toxin resistance are still suppressed by the feed of DIG-109B.t. toxin with discovery novelty.
Other insect pest that is to be understood that corn also can be tested in a similar fashion.These insects include, but are not limited to: Agromyza parvicornis (corn hybridization liriomyza bryoniae), Agrotis ipsilon (black cutworm); Anticarsia gemmatalis (velvet bean caterpillar), Diatraea grandiosella (southwestern corn borer), Diatraea saccharalis (sugarcane borer); Elasmopalpus lignosellus (lesser cornstalk borer), Helicoverpa zea (Heliothis zea), Heliothis virescens (cigarette beetle); Ostrinia nubilalis (European corn borer); Cry1F-resistance O.nubilalis, Plutella xylostella (diamond-back moth), Cry1-resistance P.xylostella; Spodoptera exigua (beet armyworm) and Trichoplusia ni (cabbage looper).
The rotaring gene corn plant that pDAS5848 transforms (T0 generation) also detects through insect bioanalysis and immunoassay.The amount of the DIG-109 albumen in the leaf extract is through commercial obtainable Cry1CELISA detection kit (Envirologix TM, Portland, MA; Cat#AP007) come quantitatively, the level of the DIG-109 albumen of detection is with a few millionths (ppm; 1ppm representes the DIG-109 albumen of the 1ng of soluble protein altogether of every mg in the extract) represent.The edible injury of FAW and rFAW is encoded as follows: 0=does not have the edible mark of injury or aperture, and the blade of 1=25%-50% is eaten, and the nearly all blade of 2=is consumed or do not have the blade residue.Protected plant is that injury values is 0.67 or lower those.
Existence and the external biological that data show in the table 9 goes out the detected DIG-109 albumen of ELISA in the T0 plant analyzed between the control of the edible injury that armyworm larvae causes in the mid-autumn has positive correlation.Plant (plant 5848-005.4) with DIG-109 albumen of highest detection level has minimum leaf feed injury score.Blade from the low-level DIG-109 of the detection albumen with 190-230ppm scope also receives edible (feeding) injury still less; Than the negative control plant (promptly; Unconverted B104 and the Hi II of contrasting) blade lock is observed, and the latter has 1.7 or 1.8 average injury score.In the pDAS5848 of all detections blade, detected main DIG-109 albumen kind comprises the about 60kDa of paired appearance and the peptide of 55kDa size.
The reduction of the level of DIG-109 albumen and the edible injury of autumn mythimna separata in the transgenic corns leaf that table 9.pDAS5848 transforms.
Figure BDA00002019672300431
aNA=is inapplicable; bThe standard deviation of SD=mean value.
Therefore the present invention has such characteristics, and the DIG-109 albumen of producing in the corn plant makes plant to the feed injury of autumn armyworm larvae and Cry1F resistance autumn armyworm larvae resistance arranged.
Embodiment 11
Produce the analysis of molecules of the corn plant of DIG-109 albumen
tissue extraction.Tissue extraction. isolation of genomic DNA the blade of the T0 rotaring gene corn plant that transforms from pDAS5162 and pDAS5848.Tissue sample be collected in 96 hole collecting boaries (Qiagen, Cat.#19560) in and freeze-drying 2 days.Klecko with embodiment 10 descriptions TMThe broken tissue of tissue powder millstone and tungsten pearl.Analyze for hydrolysis probes (HP), use DNeasy TM96 Plant kit (Qiagen) according to the flow process of manufacturer suggestion with the high throughput format isolation of genomic DNA.For the southern blotting technique analysis, with the high throughput format isolation of genomic DNA of the change method of the CTAB DNA extraction flow process of Murray and Thompson (1980).Murray，M.G.，Thompson，W.F.(1980)Rapid?isolation?of?high?molecular?weight?plant?DNA，Nucl.Acids?Res.8:4321-4325。
The DNA that extracts from any one flow process is with Quant-IT Pico Green DNA analysis kit (Molecular Probes, Invitrogen Catalog#P7589) quantification.In this step, 88 unknown samples place 96 orifice plates, and wherein first row comprise the titer of 2 times of serial dilutions, from 20ng/ μ L to 1.25ng/ μ L scope, and with the buffer solution blank, water blank and emptying aperture.The test dna sample, the 1:5 of 5 μ L and 1:40 dilution (initial concentration that depends on expectation) mix with suitable dyestuff dilution, buffer solution subsequently, in 105 μ L reaction, hatch 10 minutes under the dark.After hatching, fluorescence is with Synergy2 plate count device (BioTek, Winooski, VT) record.According to assessment genomic DNA concentration behind the calibration curve correcting background fluorescence.
The southern blotting technique preparation The 10 μ g genomic DNAs that the corn that transforms from pDAS5848 is digest down at 37 ° with Restriction Enzyme Bsm I and spend the night.The DNA sample fragment that digested through gel electrophoresis with (NC) 1% Ago-Gel separates for SAS, Cary, and be transferred to nylon membrane (INYC000I0 IMMOBILON-NY+, Millipore).Southern blotting technique and digoxigenin labeled (DIG PCR Probe Synthesis Kit; Roche Applied Science, Indianapolis, probe hybridization IN) corresponding to the pcr amplification of the 251-630 base of SEQ ID NO:8.Hybridization and detection are carried out according to supplier's flow process.The DNA from pDAS5848 conversion system that has the DIG-109 encoding gene of single copy through the southern blotting technique analysis confirmation is used as the reference that quantification PCR copy number is analyzed.
Hydrolysis probes is analyzedTransgenosis copy number by hydrolysis probes (HP) assay determination is confirmed through PCR in real time, uses
Figure BDA00002019672300441
System (Roche Applied Science).
Figure BDA00002019672300442
probe design software v 2.0 is used to design the analysis that detects following gene: DSM2 and AAD-1 selects the label gene, and (corn is sprouted appearance albumen to GLP1; GenBank Accession AY394010) and INV (corn invertase; GenBank Accession U16123) with reference to gene, and the DIG-109 encoding gene.For amplification, in 10 μ L volume mixture reactant liquors of every kind of probe of every kind of primer that contains 0.4 μ M and 0.2 μ M with 1x final concentration preparation
Figure BDA00002019672300443
Probes Master Mix (sequence of oligonucleotides and fluorescence labels is listed in table 10).Carry out 2 step amplified reactions, 56 ° are extended 40 seconds to obtain fluorescence.All samples carries out with three parts, the average Ct value every kind of sample that is used to classify.
Table 10. is used for the oligonucleotides of hydrolysis probes (HP) pcr analysis.
Figure BDA00002019672300451
aThe AAD1 probe is provided by ABI (Invitrogen)
Figure BDA00002019672300452
The MGB probe
The HP of DSM2 analyzes 36 parts of pDAS5162 conversions and fastens completion.On 95% (34 incidents) of sample, detect the simple syndication incident, be defined as 1-2 copy of gene.
The HP of AAD-1 and DIG-109 analyzes 13 parts of pDAS5848 conversions and fastens completion.In 54% (7 systems) of 93% (12 incidents) of AAD-1 sample and DIG-109, detect the simple syndication incident.These be (7 systems) 54% comprise two genes the simple syndication incident.
Embodiment 12
The biochemistry of corn DIG-109 brachymemma kind characterizes
The albumen that extracts the leaf to the T0 corn plant that transforms from pDAS5162 has carried out more detailed analysis.The Western blotting of the protein extract that carries out as probe with the DIG152RPC1 polyclonal antibody demonstrates 5 kinds of DIG-109 albumen kinds.Based on the relative movement speed of these peptides, indicate following difference: kind 1 is corresponding to DIG-109 (130kDa) albumen of the total length shown in the SEQ ID NO:5; Kind 2 is corresponding to a 70kDa DIG-109 product.In the extract of the bacterial cell of the gene of expressing coding total length DIG-152 albumen, found the peptide of same rate travel.The generation of these about 70kDa fragments indicates the main cut-out site of the full-length proteins that is exposed to the protease of all finding in corn and the bacterium.Kind 3 sizes go up the peptide corresponding to the trypsase restriction of DIG-152, and are prepared like embodiment 2, and have the size of about 65kDa; Kind 4 is corresponding to the DIG-109 product of the brachymemma of about 60kDa; Kind 5 is corresponding to the DIG-109 product of the brachymemma of about 55kDa.About 70kDa, the peptide of 60kDa and 55kDa further characterizes in embodiment 14.
Embodiment 13
The gene of design encoding D IG-109 variant and the disappearance of domain I alpha-helix
In order to promote the insecticidal properties of DIG-109 albumen, carried out disappearance continuous, progressively, remove the part of the N-end of the disclosed DIG-109 albumen of SEQ ID NO:5 at every turn.These disappearances have removed alpha-helix part or all of among the domain I 1 and part or all of alpha-helix 2, keep the structural intergrity of alpha-helix 3 to alpha-helix 7 simultaneously.Through comparing the amino acid sequence (GenBank Accession No.AAA22353) of Cry1Ca active toxin amino acid sequence and Cry1Aa albumen; We have reasoned out alpha-helix 1 among the domain I of Cry1Ca active toxin; Alpha-helix 2A; Alpha-helix 2B; The initial sum termination site of alpha-helix 3 and alpha-helix 4, and the position of the void area between them use this contrast to liking because its structure is known [RCBS Protein Structure Database Number:CRYIA (A); Grochulski et al., (1995)].These sites are described in table 1.
When the coded sequence of design N-end deletion mutant, the ATG initiation codon of a coding methionine is inserted 5 ' end of the nucleotide sequence that is designed to express said deletion mutant.For the sequence that is designed in the genetically modified plants, preferably come bonding according to Varshavsky (1997) " the N-hold-carrying then ".Its some amino acid of instruction can contribute for lability and the degraded of albumen in eukaryotic when its N-as protein holds residue.For example, it is F that the data show N-that is collected by yeast and mammalian cell holds unstable amino acid residue, L, W, Y, R, K, H, I, N, Q, D, E and possible P.Although the degradation mechanism of specific protein can be different between species, N-mentioned above holds the conservative of unstable amino acid whose homogeneity to hint that similar mechanism possibly work in plant cell.For example, Worley et al., (1998) find that the N-hold-carrying then comprises alkalescence and aromatic residues in plant.The proteolysis of plant rennet cuts off and can expose unstable n terminal amino acid near the starting point of the alpha-helix 3 of B.t. insecticidal proteins object probably.The quick decay of the protein that such process can cause cutting off and restriction B.t. insecticidal proteins build up to the level that is enough to effectively control insect.Thereby for by the initial N-end deletion mutant of unstable amino acid, the applicant preferably increases between the methionine of translation initiation and unstable amino acid and specifies the amino acid whose codon of a G (glycine).
Be described below and design disappearance.The codon optimized total length 3492 bp dna sequence dnas (that is SEQ ID NO:8) of the corn of this instance utilization coding 1164 amino acid whose chimeric DIG-109 albumen of total length (that is SEQ ID NO:5) are to illustrate the design principle of 65 specific variants.Other dna sequence dna that those skilled in the art will recognize that the whole or N-end parts of coding Cry1Ca active toxin fragment can be handled to reach expected result similarly.In order to design the first disappearance variant coded sequence, the base of all coding alpha-helixs 1 comprises all being removed near the codon of the valine (that is, the V51 of the total length DIG-109 albumen of SEQ ID NO:5) the alpha-helix 2A starting point.Thereby the deletion of 1 to 153 the base of SEQ ID NO:8 has removed the coded sequence of 1 to 51 amino acids of SEQ ID NO:5.In starting point (promptly; In front corresponding to the codon of 52 amino acids of full-length proteins) introduce a translation initiation ATG (methionine) again; This provides coded sequence for disappearance variant coded sequence; The ORFs that comprises 3342 bases, its disappearance variant (that is, methionine adds the 52-1164 amino acids of total length DIG-109 albumen) that comprises 1114 amino acid whose DIG-109 albumen of encoding.Continuous, disappearance progressively, the extra codon that it removes corresponding to the single amino acids of the residue 52-91 of the total length DIG-105 albumen of SEQ ID NO:5 provides to lack part or all of alpha-helix 2A and the variant of alpha-helix 2B.Like this, the disappearance variant coded sequence of second design requires to eliminate the base 1-156 of SEQ ID NO:8, thereby removes the coded sequence of amino acid/11-52.The recovery of functional ORFs realizes through introducing the translation initiation codon methionine again at the section start of the coded sequence that keeps once more; Thereby second deletion mutant coded sequence be provided; It has the ORFs of 3339 bases and the disappearance variant (that is, methionine adds the 53-1164 amino acids of total length DIG-109 albumen) that coding comprises 1113 amino acid whose DIG-109 albumen.The disappearance variant coded sequence of design need remove the base 1-273 of SEQ ID NO:8 at last; Eliminated the coded sequence of amino acid/11-91 like this; And, after introducing the translation initiation codon methionine again, the disappearance variant coded sequence of the ORFs with 3222 bases is provided; Its 1074 amino acid whose DIG-109 protein delation variants (that is, methionine adds the 92-1164 amino acids of total length DIG-109 albumen) of encoding.Shown in example, eliminate after the deletion sequence, add an initial codon methionine with the restore functionality ORFs in the starting point of the coded sequence that keeps.As previously mentioned; Extra codon glycine will be added between the amino acid whose codon that codon methionine and lability confirm, what expose with the N-end of the reserve part of the full-length proteins that prevents deletion sequence is the amino acid that a lability that preamble was provided is confirmed.
Table 11 has been described the specific variants according to above-described strategy design.
The disappearance misfolded proteins sequence of the total length DIG-109 albumen of table 11.SEQ ID NO:5.
Figure BDA00002019672300481
Figure BDA00002019672300491
Other nucleic acid of the DIG-109 protein variant of describing in the coding schedule 11 designs according to the conventional principles of the synthetic property gene that tends to express in the plant, like 6 instructions of embodiment.
Embodiment 14
Design other DIG-109 variant
Disclosed like embodiment 12, the initial translation product that comprises total length DIG-109 albumen is processed to various degree in plant, and one of them product is corresponding to the active toxin peptide of the trypsase brachymemma of 65kDa.This active toxin is considered to combine the acceptor of insect midgut and the activity form of the toxin that causes poisoning.Trypsase is endopeptidase, and it is at the distolateral scinderin of C-of arginine (R) or lysine (K) residue.Therefore, observed 65kDa DIG-109 peptide maybe be corresponding to by the trypsase albuminoid enzyme of the corn 65kDa fragment that cutting is produced after the residue R28 of SEQ ID NO:5 and R628 in the corn.Notice that this 65kDa active toxin peptide possibly comprise the amino acid/11-9 of Cry1Ab parent toxin fragment of amino acid 28-619 and SEQ ID NO:4 of the Cry1Ca active toxin fragment of SEQ ID NO:1.But, should be appreciated that the brachymemma product of 65kDa, or the accurate C-end of other brachymemma product of in transgenic corns, observing and discussing hereinafter, and without verification experimental verification.Therefore, it is schematically that the design of the DIG-109 misfolded proteins of this paper discussion is attempted, and other DIG-109 truncated variant albumen of reservation insecticidal activity also within the scope of the invention.
The concentration of the DIG-109 peptide prod that shows in most of rotaring gene corn plants is confirmed as about 200ppm.Therefore, can and confirm that the material of the amino acid sequence of these DIG-109 peptides is insufficient from the manual purifying of plant tissue.Through use different protease cut off total length DIG-152 albumen produced with corn in the truncated protein that detects similarly substitute peptide in size.
The identity of 70kDa peptide.The SDS-PAGE diagram of the total length DIG-152 that produces as inclusion body in the pseudomonas fluorescens (Pf) shows that a large amount of albumen have the molecular weight that seems 70kDa, and is metastable to trypsase.After the purifying, this peptide has the identical mobility of DIG-109 peptide with detected about 70kDa from the rotaring gene corn plant extract on SDS-PAGE from the total length DIG-152 inclusion body of dissolving in combination through anion exchange and size exclusion chromatogram.Two kinds of peptides are all by the polyclonal antibody identification to DIG-152, and the amino acid analysis of the peptide of Pf-production identifies MDNNP as its N-terminal sequence (DIG-109, the residue 1-5 of SEQ ID NO:5).Therefore this 70kDa peptide contains self N-end of total length DIG-109 albumen.Trypsase is being inferred the cut-out that site (R628) cut in active toxin C-end-grain cutting; Stay complete 28 residues at first simultaneously; Thereby being removed from DIG-109 albumen on the shut-off features property ground of R28 position through trypsase, the latter produces active toxin; Produced the peptide that the size with calculating is 70.5kDa (the residue 1-628 that comprises DIG-109), with from the Pf inclusion bodies separating and rotaring gene corn plant detected DIG-152 peptide almost can not distinguish.Thereby the identity of this 70kDa albumen is considered to the DIG-109 peptide corresponding to the brachymemma that comprises amino acid/11-628.
The identity of 60dDa and 55kDa peptide.Find that the corn plant that pDAS5162 and pDAS5848 transform also produces the DIG-109 derived protein corresponding to the mobility of 60kDa and 55kDa.These big or small peptides produce with the product that chymotrypsin processing trypsase cuts off through at first cutting off total length DIG-152 albumen with trypsase experimentally then.[total length DIG-152 albumen causes the multiple brachymemma product less times greater than 60kDa with the chymotrypsin individual processing.] prepare in a large number trypsase/chymotrypsin cut off product then through anion-exchange chromatography then Superose 200 size exclusion chromatograms come purifying.In the size exclusion chromatographic step, observe 3 main peaks, at 12.5mL, 18.3mL and 20mL collected volume place wash-out.First main peaks (12.5mL) comprises the DIG-152 albumen set of HMW (700kDa to 1000kDa), and the 3rd main peaks (20mL) comprises excessive chymotrypsin.12.5mL part also contains such band, it has the mobility corresponding to the 65kDa of DIG-152 and 60kDa product; Therefore the oligomerization that demonstrates the DIG-152 derived peptide is reversible with assembling.
18.3mL the albumen at peak with the DIG-152 albumen that trypsase only cuts off, is analyzed under reduction and sex change condition through SDS-PAGE.These albumen comprise two kinds of main kinds, have the mobility corresponding to 60kDa and 55kDa.As if also observe the albumen of littler 14kDa and 9kDa and differentiate to be chymotrypsin, it is being bonded to during purifying on the DIG-152 peptide.In addition, observe the HMW band that has corresponding to the mobility of 240kDa.The albumen of this band is proved its most likely oligomer (tetramer) of DIG-152 cut-out product by the DIG152RPC1 antibody recognition.
Separate the albumen in the extract that DIG-109 produces plant through SDS-PAGE, then electroblotting to nitrocellulose, with purification of samples, the DIG-152 that trypsase cuts off and with the trypsase DIG-152 albumen that cuts off of chymotrypsin then.Through elementary DIG152RPC1 rabbit antibody and secondary anti--enhanced chemiluminescence that the antibody of rabbit horseradish peroxidase-labeled causes demonstrates the band corresponding to DIG-109 or DIG-152 peptide.The DIG-152 sample of trypsin treatment shows single band at about 65kDa mobility place.The extract of producing plant from the DIG-109 peptide shows 4 bands: the mobility corresponding to 130kDa, (representing total length DIG-109 albumen) is corresponding to band and band corresponding to the mobility of about 20kDa of the mobility of 60kDa and 55kDa.There is not further to characterize the cut-out product of the 20kDa of DIG-109.With trypsase then the DIG-152 albumen handled of chymotrypsin show 2 bands, have mobility corresponding to about 60kDa and 55kDa, observed 60kDa and 55kDa are mobile in plant extracts.The band of a macromolecule is also arranged in the trypsase DIG-152 protein sample that chymotrypsin is handled then, have mobility corresponding to about 240kDa.
Therefore, the main cut-out product of the DIG-109 that produces in the corn at first cuts off, further cuts off 2 products that the back obtains with chymotrypsin then with trypsase corresponding to total length DIG-152 albumen in size.5 N-at first of 60kDa that enzyme handle to produce and 55kDa peptide hold residue all to be confirmed as DAFLV (corresponding to the residue 74-78 of the DIG-109 albumen of SEQ ID NO:5).Notice that the cut-out of total length DIG-109 albumen after W73 causes having removed alpha-helix 1, the alpha-helix 2B (table 1) of alpha-helix 2A and part.
Notice that further because 60kDa and 55kDa peptide all have same N-terminal sequence, the 5kDa fragment that in the generation of less (55kDa) peptide, is removed is necessarily being represented terminal further the removing of C-of 60kDa peptide.
The amino acid coordinate of inferring of 5 kinds of main DIG-109 peptides that the corn plant that is transformed by pDAS5162 and pDAS5848 produces is summarised in table 12.The accurate C-end of these kinds is not confirmed.The kind 4 of noticing 60kDa is cut off the peptide that will produce 56kDa, (that is, approaching with kind 5) through trypsase after R568.
Table 12. is by the identity of inferring of the protein derived processed peptide of DIG-109 and DIG-152.Inferred C-end position roughly according to the roughly molecular weight of gel.In amino acid no is included in.
DIG-109 or DIG-152 peptide The residue of SEQ ID NO:5
Kind 1 (130kDa) 1-1164 (MW 131.7 of calculating)
Kind 2 (70kDa) 1-628 (MW 70.59 of calculating)
Kind 3 (the core that trypsase produces; 65kDa) 28-628 (600 residues, the MW 67.4 of calculating)
Kind 4 (60kDa) 74-628 (555 residues, the MW 62.7 of calculating)
Kind 5 (55kDa) 74-568 (495 residues, the MW 56.1 of calculating)
design DIG-109 truncated variant.Such as in the table 1 displaying, alpha-helix 1 to the alpha-helix 4 of DIG-109 active toxin is present in 145 amino acid residues at first of DIG-109 albumen.Cut-out (the R87 of DIG-109 in first potential site that the N-of DIG-109 active toxin is terminal; The R39 of active toxin) will from the DIG-109 core, remove 59 amino acid, and produce the albumen with 61.02kDa molecular weight, and have alpha-helix 1, alpha-helix 2A and alpha-helix 2B remove.The removing of the alpha-helix 1 of Cry1Ab involves in allowing albumen initially to be incorporated into the bypass of cadherin acceptor, cause forming oligomerization before-core texture, insert the insect midgut cell membrane afterwards and finally cause the formation in hole.According to similar research, prediction removes the N-end parts of the DIG-109 core of trypsase brachymemma, causes losing alpha-helix 1, is that the permission oligomer forms and is bonded to secondary Aminopeptidase N acceptor and causes the formation in functional hole needed.Therefore, DIG-109 albumen cuts off the bypass that can cause being provided by the DIG-109 toxin peptide of insect picked-up the demand that is bonded to the cadherin acceptor by this way in the plant.Such effect has shown the overcoming property resistance of in the insect of the cadherin receptor protein that causes having variation Bt albumen being poisoned.
The less peptide of finding in pDAS5162 and the pDAS5848 rotaring gene corn plant (60kDa and 55kDa) possibly represented the product of the further cut-out of trypsase albuminoid enzyme.Because these peptides are only than the little 5kDa to 10kDa of core peptide of 65kDa, so further cut-out can remove and be less than about altogether 80 residues from any a section of active toxin.Within 130 residues at first of the N-of DIG-109 albumen end; Potential trypsase cuts off the site and is positioned at R28 (R-1 of active toxin), R87 (R59 of active toxin), R93 (R65 of active toxin); K115 (K87 of active toxin); K122 (K94 of active toxin), R127 (R99 of active toxin), and R129 (R101 of active toxin).Within last 100 residues of the C-of active toxin end; Potential trypsase cuts off the site and is positioned at R530 (R502 of active toxin), R533 (R505 of active toxin), K557 (K529 of active toxin); R568 (R540 of active toxin); R571 (R543 of active toxin), R582 (R554 of active toxin), and K610 (K582 of active toxin).
The position in potential protease cutting site that utilizes above-mentioned evaluation is as guiding, and the dna sequence dna that the DIG-109 coded sequence of the disclosed corn optimization of SEQ ID NO:8 is derived is designed to the DIG-109 protein variant of genetic coding brachymemma.Embodiment 13 disclosed guilding principles of before the coded sequence of brachymemma, adding 5 ' end methionine and codon glycine are still used in these structures are carried.The embodiment that first is such, DIG-110, disclosed like SEQ ID NO:27, comprise the amino acid 88-1164 of DIG-109 albumen and methionine and the glycine that the N-end adds.The dna sequence dna that the corn of encoding D IG-110 is optimized is open as SEQ ID NO:28.Second embodiment, DIG-111, disclosed like SEQ ID NO:29, comprise the amino acid 88-628 of DIG-109 albumen and methionine and the glycine that the N-end adds.The dna sequence dna that the corn of encoding D IG-111 is optimized is open as SEQ ID NO:30.The 3rd embodiment, DIG-112, disclosed like SEQ ID NO:31, comprise the amino acid/11 23-1164 of DIG-109 albumen and methionine and the glycine that the N-end adds.The dna sequence dna that the corn of encoding D IG-112 is optimized is open as SEQ ID NO:32.The 4th embodiment, DIG-113, disclosed like SEQ ID NO:33, comprise the amino acid/11 23-628 of DIG-109 albumen and methionine and the glycine that the N-end adds.The dna sequence dna that the corn of encoding D IG-113 is optimized is open as SEQ ID NO:34.The 5th embodiment, DIG-114, disclosed like SEQ ID NO:35, comprise the amino acid/11-582 of DIG-109 albumen.The dna sequence dna that the corn of encoding D IG-114 is optimized is open as SEQ ID NO:36.
It should be noted that DIG-110 and DIG-112 albumen comprise the disclosed Cry1Ab parent toxin of SEQ ID NO:4 fragment.Make protein stable or make its soluble function in plant according to thinking that this C-end parent toxin fragment possibly play in some cases.The cut-out in the R543 trypsase site of DIG-110; Thereby remove the major part of parent toxin fragment; With producing calculated size is the peptide of 61.2kDa, and observed 60kDa DIG-109 truncated peptide is very approaching in the corn plant that this size and pDAS5162 and pDAS5848 transform.DIG-111 albumen (it lacks all Cry1Ab parent toxin fragments except at first 9 amino acid) comprises the fragment that the ability of DIG-110 produces through such cut-out (that is, the amino acid/11 of DIG-110-543, calculated size is 61.2kDa).
Similarly, the cut-out in the similar R508 site of DIG-112 will produce the peptide that calculated size is 57.2kDa, and observed 55kDaDIG-109 truncated peptide is very approaching in the corn plant that this size and pDAS5162 and pDAS5848 transform.DIG-113 albumen (it lacks all Cry1Ab parent toxin fragments except at first 9 amino acid) comprises the fragment that the ability of DIG-112 produces through such cut-out (that is, the amino acid/11 of DIG-112-508, calculated size is 57.2kDa).
DIG-114 albumen kept DIG-109 albumen amino acid/11-28 (these residues can in plant or in insect midgut enzyme remove) and and the potential trypsase that ends at the R582 place of DIG-109 albumen cut off the site.Thereby this DIG-109 variant can exist with 65.7kDa albumen or 62.2 peptides, depends on whether 28 amino acid of N-end are removed in the body.
The coded sequence that has designed extra corn optimization through the disclosed principle of this paper is with the further DIG-109 protein variant of encoding.
Embodiment 15
The expression plasmid of structure encoding D IG-109 and DIG-109 misfolded proteins is also shown in pseudomonad Reach
The standard cloning process [is described in for example Sambrook et al., (1989) and Ausubel et al., (1995); And upgrade] be used to make up pseudomonas fluorescens (Pf) expression construct; The latter is processed into and produces DIG-109 albumen or DIG-110, DIG-111, DIG-112; DIG-113, or DIG-114 albumen (being referred to as the DIG-109 misfolded proteins).Protein expression is at the pseudomonas fluorescens MB214 (derivative of bacterial strain MB101; The biovar I of pseudomonas fluorescens) carry out in, this bacterial strain has the lac operon of the modification of insertion, like 5169760 descriptions of the US patent No..Basic clone's strategy comprises that the dna fragmentation of subclone encoding D IG-109 or DIG-109 misfolded proteins is to plasmid pDOW1169; And place (PL Pharmacia from plasmid pKK223-3; Milwaukee is under Ptac promotor WI) and the expression of the rrnBT1T2 terminator control.PDOW1169 is medium copy plasmid; Has the RSF1010 replication origin, pyrF gene, and ribosome bind site; Be the Restriction Enzyme recognition site afterwards, the dna fragmentation that contains the encoding histone zone can be introduced into wherein (US number of patent application 20080193974).This expression is administered (near the pseudomonas fluorescens strain of wild type, is had sudden change Δ pyrF and lsc::lacI through electroporation conversion entering DC454 QI), or derivatives thereof is recovered in SOC-soybean hydrolysate medium, be tiled in select medium (the M9 agar glucose lacks uracil, Sambrook et al., supra).The details of microbiology operation is referring to Squires et al., and (2004), US number of patent application 20060008877, US number of patent application 20080193974 and US number of patent application 20080058262 are incorporated this paper into as a reference.The clone at first screens through PCR, and positive colony is analyzed through restrictive diges-tion a small amount of DNA subsequently.The selected clone's who comprises insert DNA through the commercial order-checking of contact supplier for example MWG Biotech (Huntsville AL) checks order.Use Sequencher TMSoftware (Gene Codes Corp., Ann Arbor, MI) assembling and analytical sequence.
Shake growth and expression analysis in the bottleBe used to characterize with the DIG-109 albumen of insect bioanalysis or the production of DIG-109 misfolded proteins and accomplish through the pseudomonas fluorescens strain that comprises suitable expression plasmid that shakes the bottle growth.The production of DIG-109 albumen or DIG-109 misfolded proteins is by the Ptac promoters driven, and as the previous US patent No. 5527883 as described in operate.Shake 30 ℃ of initial cultivations of bottle and come abduction delivering through adding isopropyl-β-D-1-thiogalactoside (IPTG) after 24 hours.Culture is when inducing and induce the back different time to take a sample.Optical density (OD600) through at 600nm is measured cell density.At each time point, the cell density of sample is adjusted to OD 600=20,1mL component under 14000xg centrifugal 5 minutes.The cell fritter is freezing down at-80 °.
Embodiment 16
The cell of the shake-flask culture sample of the pseudomonad of production DIG-109 and DIG-109 misfolded proteins Branch and SDS-PAGE analyze
Solvable and insoluble part from freezing shake-flask culture cell fritter sample is used EasyLyse TMBacterioprotein extraction solution (
Figure BDA00002019672300541
Biotechnologies, Madison WI) produces.Use embodiment 2 disclosed methods and guilding principle.
Embodiment 17
The insecticidal activity of the DIG-109 misfolded proteins of producing in the pseudomonas fluorescens
The insecticidal activity of DIG-109 misfolded proteins is verified on the Lepidoptera species, comprises European corn borer (ECB; Ostrinia nubilalis (H ü bner)), cry1F-resistance ECB (rECB), cotton bollworm (CEW; Helicoverpa zea (Boddie)), black cutworm (BCW; Agrotis ipsilon (Hufnagel)), autumn mythimna separata (FAW, Spodoptera frugiperda (J.E.Smith)), Cry1F-resistance FAW (rFAW), southwestern corn borer (SWCB, Diatraea grandiosella), sucrose snout moth's larva (SCB; Diatraea saccharalis) and Cry1Ab-resistance SCB (rSCB).
Deferring to embodiment 3 carries out with embodiment 4 disclosed methods, guilding principle and data.
Embodiment 18
The structure of plant conversion carrier that comprises the expressive gene of plant of encoding D IG-109 misfolded proteins
(Japan Tobacco, Tokyo JP) are used for monocotyledon host's conversion to the super double element system of Agrobacterium expediently.The structure of plant expression vector, and the generation of super double base plasmid is confirmed to carry out with embodiment 8 disclosed methods through embodiment 7 with it.PSB11 derive plasmid the T-DNA component the concise and to the point signal of physical arrangement as follows:
RB>corn Ubi1 promotor: DIG-109 variant CDS: corn Per5 3'UTR>paddy rice Act1 promotor: DSM2 CDS: corn Lip 3'UTR>LB, or
RB>corn Ubi1 promotor: DIG-109 variant CDS: corn Per5 3'UTR>corn Ubi1 promotor: AAD-1 CDS: corn Lip 3'UTR>LB
Embodiment 19
Produce the DIG-109 protein variant in the corn plant
The conversion of agriculture bacillus mediated cornThe rotaring gene corn plant of producing the DIG109 misfolded proteins produces through embodiment 9 disclosed methods.
The technical staff in corn conversion field will understand other method corn conversion and screening conversion plant also will be fine, when using the effable selected marker of other plant (for example herbicide tolerant gene).
Embodiment 20
The biochemistry and the branch of the rotaring gene corn plant of the gene of expression encoding D IG-109 misfolded proteins Son is analyzed and the insect bioanalysis
The biochemistry of DIG-109 misfolded proteins of rotaring gene corn plant production that has and express the gene of encoding D IG-109 misfolded proteins characterizes through method and the reagent of embodiment 10 and embodiment 12 operates.The transgenic analysis of the gene of encoding D IG-109 misfolded proteins is carried out according to embodiment 11 disclosed methods and reagent.Being derived from the insect bioanalysis of blade of rotaring gene corn plant that has and express the gene of encoding D IG-109 misfolded proteins operates through embodiment 10 disclosed methods.
Figure IDA00002019672700011
Figure IDA00002019672700021
Figure IDA00002019672700031
Figure IDA00002019672700041
Figure IDA00002019672700051
Figure IDA00002019672700061
Figure IDA00002019672700071
Figure IDA00002019672700091
Figure IDA00002019672700101
Figure IDA00002019672700111
Figure IDA00002019672700121
Figure IDA00002019672700131
Figure IDA00002019672700161
Figure IDA00002019672700171
Figure IDA00002019672700181
Figure IDA00002019672700191
Figure IDA00002019672700201
Figure IDA00002019672700211
Figure IDA00002019672700221
Figure IDA00002019672700241
Figure IDA00002019672700251
Figure IDA00002019672700261
Figure IDA00002019672700271
Figure IDA00002019672700281
Figure IDA00002019672700291
Figure IDA00002019672700301
Figure IDA00002019672700311
Figure IDA00002019672700321
Figure IDA00002019672700331
Figure IDA00002019672700351
Figure IDA00002019672700361
Figure IDA00002019672700371

Claims (23)

1. Cry1Ca misfolded proteins with insecticidal activity, wherein corresponding to the N-end alpha-helix 1 of wild type Cyr1Ca, 2A, and/or all or part of 2B is deleted.
2. the misfolded proteins of claim 1, deletion have wherein removed whole and alpha-helix 2 all or part of of alpha-helix 1 among the domain I, and said albumen comprises alpha-helix 3 to 7.
3. the misfolded proteins of claim 1, wherein said deletion has improved the insecticidal activity of insecticidal proteins DIG-109, and but wherein said deletion originates in before the alpha-helix 2A starting point and ends at after the alpha-helix 2B terminal point do not extend in the alpha-helix 3.
4. the misfolded proteins of claim 1, wherein said deletion has improved the insecticidal activity of insecticidal proteins DIG-152, and but wherein said deletion originates in before the alpha-helix 2A starting point and ends at after the alpha-helix 2B terminal point do not extend in the alpha-helix 3.
5. the misfolded proteins of claim 1; Wherein the deletion of N-end begins with at least one unstable amino acid; And said albumen comprises the codon of interpolation, and this codon is prescribed coding (specify) glycine between the methionine of translation initiation and said unstable amino acid.
6. the misfolded proteins of claim 1, wherein said albumen lack C-end parent toxin sequence.
7. the Cry1Ca albumen of modifying is included in the protease cutting site of introducing in the spacer region between alpha-helix 2B and the alpha-helix 3.
8. the albumen of the modification of claim 7, wherein said protease cutting site are introduced in the amino acid 85-90 of Cry1Ca active toxin albumen of SEQ ID NO:1.
9. claim 1 or 7 albumen, wherein said albumen has improved activity to insect.
10. the albumen of claim 9, wherein said insect are selected from autumn mythimna separata and sugarcane borer (sugarcane borer).
11. the Cyr1Ca albumen of modifying, comprise exchange or reset/domain that is selected from domain II and domain II I of reorganization.
12. the albumen of claim 1 comprises the active toxin fragment, this fragment comprises the amino acid/11-619 of SEQ ID NO:1, wherein has 10 of as many as, 15 of as many as, or 20 amino acid whose interpolations of as many as, deletion or replacement.
13. the insecticidal proteins of a fusion comprises the N-end core toxin fragment that is derived from the Cry1Ca insecticidal proteins, merge in itself and the allos δNei Dusu parent toxin fragment site after said active toxin fragment.
14. the albumen of claim 12, said albumen comprise the amino acid sequence of the residue 28-619 of SEQ ID NO:1, and have 20 amino acid whose replacements of as many as, deletion or modification.
15. the albumen of claim 12, said albumen comprise the residue 1-619 of SEQ ID NO:1, and have 20 amino acid whose replacements at the most, deletion or modification.
16. the albumen that separates, its be selected from SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, the sequence of SEQ ID NO:35 and SEQ IDNO:1 has at least 90% homogeneity.
17. comprising, the albumen of claim 16, wherein said albumen is selected from SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, the sequence of SEQID NO:35 and SEQ ID NO:1.
18. the polynucleotides that separate, the albumen of its coding claim 16.
19. the polynucleotides of claim 18, wherein said polynucleotides be selected from SEQ ID NO:4, SEQ ID NO:8; SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32; SEQ IDNO:34, the sequence of SEQ ID NO:36 and SEQ ID NO:2 has at least 90% homogeneity.
20. the method for control lepidopterous insects, said method comprise the albumen that makes said insect contact claim 16.
21. produce the plant cell of the albumen of claim 16.
22. comprise the microbial cell of the polynucleotides of claim 18.
23. comprise the plant of the cell of a plurality of claims 21.
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